Nanoparticle complexes of paclitaxel, anti-cd52 antibodies, and albumin

ABSTRACT

Described are nanoparticle complexes comprising paclitaxel, albumin and anti-CD52 antibody. The nanoparticle complexes are suitable for the treatment of cancer, in particular cancer that expresses CD52.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/708,575, filed Oct. 1, 2012, and U.S. Provisional ApplicationSer. No. 61/725,293, filed Nov. 12, 2012. The disclosure of the priorapplications are considered part of (and are incorporated by referencein) the disclosure of this application.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in treatingcancer (e.g., skin cancers such as melanoma). For example, this documentrelates to methods and materials involved in using complexes containingalbumin-containing nanoparticles (e.g., ABRAXANE® nanoparticles) andantibodies (e.g., anti-VEGF polypeptide antibodies such as AVASTIN®) totreat cancer. This document also relates to methods and materialsinvolved in using ABRAXANE® in combination with an anti-VEGF polypeptideantibody (e.g., AVASTIN®) to treat skin cancer.

2. Background Information

Melanoma is the most serious form of skin cancer. It is a malignanttumor that originates in melanocytes, the cells which produce thepigment melanin that colors skin, hair, and eyes and is heavilyconcentrated in most moles. While it is not the most common type of skincancer, melanoma underlies the majority of skin cancer-related deaths.About 48,000 deaths worldwide are registered annually as being due tomalignant melanoma. Worldwide, there are about 160,000 new cases ofmelanoma each year. Melanoma is more frequent in white men and isparticularly common in white populations living in sunny climates. Otherrisk factors for developing melanoma include a history of sunburn,excessive sun exposure, living in a sunny climate or at high altitude,having many moles or large moles, and a family or personal history ofskin cancer.

Melanomas fall into four major categories. Superficial spreadingmelanoma can travel along the top layer of the skin before penetratingmore deeply. Lentigo maligna typically appears as a flat or mildlyelevated mottled tan, brown, or dark brown discoloration and is foundmost often in the elderly. Nodular melanoma can occur anywhere on thebody as a dark, protuberant papule or a plaque that varies from pearl togray to black. Acral-lentiginous melanoma, although uncommon, is themost common form of melanoma in blacks. It can arise on palmar, plantar,or subungual skin. Metastasis of melanoma occurs via lymphatics andblood vessels. Local metastasis results in the formation of nearbysatellite papules or nodules that may or may not be pigmented. Directmetastasis to skin or internal organs can occur.

SUMMARY

This document provides methods and materials involved in treating cancer(e.g., skin cancers such as melanoma). For example, this documentprovides methods and materials for using complexes containingalbumin-containing nanoparticles (e.g., ABRAXANE® nanoparticles) andantibodies (e.g., anti-VEGF polypeptide antibodies such as AVASTIN®) totreat cancer. This document also provides methods and materials involvedin using ABRAXANE® in combination with an anti-VEGF polypeptide antibody(e.g., AVASTIN®) to treat skin cancer (e.g., melanoma). ABRAXANE® isavailable from Celgene Corp. and is a nanoparticle formulation thatcombines paclitaxel with human albumin. AVASTIN® is also known asbevacizumab and is available from Genentech Corp. and Roche Corp.AVASTIN® is a humanized monoclonal antibody that binds to vascularendothelial growth factor A. As described herein, in vitro mixing ofalbumin-containing nanoparticles (e.g., ABRAXANE® nanoparticles) andantibodies (e.g., bevacizumab, bevacizumab, trastuzamab, or rituxan) canresult in the formation of macromolecular complexes, the characteristicsof which (e.g., size, antibody content, or chemotherapeutic drugcontent) can be customized depending on need. In some cases, suchmacromolecular complexes can retain antibody mediated target bindingspecificity, can retain or exhibit enhanced chemotherapeutic tumor cellcytotoxicity, and can exhibit no additional toxicity beyond that ofABRAXANE® nanoparticles alone. As also described herein, contactingABRAXANE® with an anti-VEGF polypeptide antibody (e.g., AVASTIN®) priorto administration to a human (e.g., a human melanoma cancer patient) canresult in a complex that, when administered as a complex, has anincreased ability to treat melanoma as compared to a treatment regimenthat includes administering ABRAXANE® and the anti-VEGF polypeptideantibody separately in a manner that does not form ABRAXANE®/anti-VEGFpolypeptide antibody complexes.

The methods and materials provided herein can be used to increase theprogression-free survival rate in skin cancer patients. Increasingprogression-free survival can allow skin cancer patients to live longer.

In general, one aspect of this document features a method for treating amammal having skin cancer. The method comprises, or consists essentiallyof, administering to the mammal a composition containingABRAXANE®/anti-VEGF polypeptide antibody complexes (or complexes of (a)an anti-VEGF polypeptide antibody and (b) human albumin-containingnanoparticles having an agent other than placitaxel) under conditionswherein the length of progression-free survival is increased. The mammalcan be a human. The skin cancer can be melanoma. The skin cancer can bestage IV melanoma. In some cases, a composition comprisingABRAXANE®/AVASTIN® complexes can be administered to the mammal. Thecomposition can comprise an alkylating agent. The alkylating agent canbe a platinum compound. The platinum compound can be carboplatin. Theanti-VEGF polypeptide antibody can be a humanized antibody. Theanti-VEGF polypeptide antibody can be bevacizumab. The composition canbe administered by injection. The progression-free survival can beincreased by 25 percent. The progression-free survival can be increasedby 50 percent. The progression-free survival is increased by 75 percent.The progression-free survival can be increased by 100 percent. Thecomposition can be administered under conditions wherein the time toprogression is increased.

In another aspect, this document features a method for treating a mammalhaving cancer. The method comprises, or consists essentially of,administering, to the mammal, a composition comprisingalbumin-containing nanoparticle/antibody complexes, wherein the averagediameter of the complexes is between 0.1 and 0.9 μm. The mammal can be ahuman. The cancer can be skin cancer. The skin cancer can be melanoma.The skin cancer can be stage IV melanoma. The albumin-containingnanoparticle/antibody complexes can be ABRAXANE®/AVASTIN® complexes. Thecomposition or the albumin-containing nanopartiele/antibody complexescan comprise an alkylating agent. The alkylating agent can be a platinumcompound. The platinum compound can be carboplatin. The antibodies ofthe albumin-containing nanoparticle/antibody complexes can be anti-VEGFpolypeptide antibodies. The anti-VEGF polypeptide antibodies can behumanized antibodies. The anti-VEGF polypeptide antibodies can bebevacizumab. The composition can be administered by injection. Theadministration of the composition can be effective to increaseprogression-free survival by 25 percent. The administration of thecomposition can be effective to increase progression-free survival by 50percent. The administration of the composition can be effective toincrease progression-free survival by 75 percent. The administration ofthe composition can be effective to increase progression-free survivalby 100 percent. The administration of the composition can be underconditions wherein the median time to progression for a population ofmammals with the cancer is at least 150 days. The administration of thecomposition can be under conditions wherein the median time toprogression for a population of mammals with the cancer is at least 165days. The administration of the composition can be under conditionswherein the median time to progression for a population of mammals withthe cancer is at least 170 days. The average diameter of the complexescan be from 0.1 μm to 0.3 μm. The average diameter of the complexes canbe from 0.15 μm to 0.3 μm. The average diameter of the complexes can befrom 0.2 μm to 0.5 μm. The average diameter of the complexes can be from0.3 μm to 0.5 μm. The average diameter of the complexes can be from 0.2μm to 0.8 μm. The average diameter of the complexes can be from 0.2 μmto 0.7 μm.

In another aspect, this document features a method for treating a mammalhaving cancer. The method comprises, or consists essentially of,administering, to the mammal, a composition comprisingalbumin-containing nanoparticle/antibody complexes, wherein the averagediameter of at least 5 percent of the complexes of the composition isbetween 0.1 and 0.9 μm. The mammal can be a human. The cancer can beskin cancer. The skin cancer can be melanoma. The skin cancer can bestage IV melanoma. The albumin-containing nanoparticle/antibodycomplexes can be ABRAXANE /AVASTIN® complexes. The composition or thealbumin-containing nanoparticle/antibody complexes can comprise analkylating agent. The alkylating agent can be a platinum compound. Theplatinum compound can be carboplatin. The antibodies of thealbumin-containing nanoparticle/antibody complexes can be anti-VEGFpolypeptide antibodies. The anti-VEGF polypeptide antibodies can behumanized antibodies. The anti-VEGF polypeptide antibodies can bebevacizumab. The composition can be administered by injection. Theadministration of the composition can be effective to increaseprogression-free survival by 25 percent. The administration of thecomposition can be effective to increase progression-free survival by 50percent. The administration of the composition can be effective toincrease progression-free survival by 75 percent. The administration ofthe composition can be effective to increase progression-free survivalby 100 percent. The administration of the composition can be underconditions wherein the median time to progression for a population ofmammals with the cancer is at least 150 days. The administration of thecomposition can be under conditions wherein the median time toprogression for a population of mammals with the cancer is at least 165days. The administration of the composition can be under conditionswherein the median time to progression for a population of mammals withthe cancer is at least 170 days. The average diameter of at least 5percent of the complexes of the composition can be from 0.2 μm to 0.9μm. The average diameter of at least 5 percent of the complexes of thecomposition can be from 0.2 μm to 0.8 μm. The average diameter of atleast 5 percent of the complexes of the composition can be from 0.2 μmto 0.7 μm. The average diameter of at least 5 percent of the complexesof the composition can be from 0.2 μm to 0.6 μm. The average diameter ofat least 5 percent of the complexes of the composition can be from 0.2μm on to 0.5 μm. The average diameter of at least 5 percent of thecomplexes of the composition can be from 0.2 μm to 0.4 μm. The averagediameter of at least 10 percent of the complexes of the composition canbe between 0.1 and 0.9 μm. The average diameter of at least 50 percentof the complexes of the composition can be between 0.1 and 0.9 μm. Theaverage diameter of at least 75 percent of the complexes of thecomposition can be between 0.1 and 0.9 μm. The average diameter of atleast 90 percent of the complexes of the composition can be between 0.1and 0.9 μm.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention arc set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram of an ABRAXANE® nanoparticle (labeled A) complexedwith an anti-VEGF polypeptide antibody (bevacizumab; labeled B). In twoof the three cases, the anti-VEGF polypeptide antibody is shown bindingto a VEGF-A polypeptide (labeled V), and a fluorescently-labeledanti-VEGF antibody (labeled aV*) is shown bound to the VEGF-Apolypeptide.

FIG. 2 contains scatter plots of a flow cytometry analysis plotting thelevel of yellow fluorescence of A alone, A plus aV*, A plus B plus aV*,A plus V plus aV*, or A plus B plus V plus aV*. The labels are asindicated in FIG. 1. These results demonstrate that A and Bspontaneously associate and preserve a VEGF polypeptide bindingpotential.

FIG. 3 is graph that contains the flow cytometry data from FIG. 2.

FIG. 4 is a repeat of the experiment of FIG. 3, comparing A alone, Aplus aV*, A plus B plus aV*, A plus V plus aV*, or A plus B plus V plusaV*. One difference is in FIG. 3, 500 ng of VEGF was used. In FIG. 4,100 ng VEGF was used to visualize the complex.

FIG. 5 is a graph plotting flow cytometry data of A plus B incubated inthe presence of various concentrations of human plasma (1:1 to 1:16)followed by addition of V and aV*. These results indicate that humanplasma diluted in a range of relative volumes (1:1 to 1:16) successfullyinhibited the formation of the A+B complex relative to controls.

FIG. 6 is a graph plotting flow cytometry data of A plus B incubated inthe presence of various concentrations of human serum albumin (500 μg,50 μg, 5 μg, 0.5 μg, and 0.05 μg/mL) followed by addition of V and aV*.These results indicate that incubation with serum albumin(concentrations ranging from 500 μg/mL to 0.05 μg/mL) did not affect thecomplexing of A and B.

FIG. 7 is a graph plotting flow cytometry data of A plus B incubated inthe presence of various concentrations of human polyclonalimmunoglobulin (500 μg, 50 μg, 5 μg, 0.5 μg, and 0.05 μg/mL) followed byaddition of V and aV*. These results indicate that incubation of A and Bwith a range of concentrations of human immunoglobulin (IVIG; 500 mg/mLto 0.05 μg/mL) partially inhibited A and B complexing.

FIG. 8 contain A plus B complexing results in the presence of plasma(1:1), WIG (0.5 mg/mL), or albumin (0.5 mg/mL). At the highestconcentrations of plasma (1:1), IVIG (0.5 mg/mL), or albumin (0.5mg/mL)tested, the levels of relative inhibition of A plus B complexing differin diminishing order.

FIG. 9 contains photographs of light microscope images of ABRAXANE®(ABX) or mixtures of ABRAXANE® (ABX) and bevacizumab (BEV; 0.5, 5, 10,or 25 mg/mL) either 4 or 24 hours after mixing.

FIG. 10 is a graph plotting flow cytometry results of ABRAXANE® alone,ABX:BEV complexes, and 2 μm standard beads.

FIG. 11 is graph plotting the proliferation index for A375 cells (amelanoma tumor cell line) exposed to ABRAXANE® (ABX) only,ABRAXANE®:Herceptin (non-VEGF targeting) complexes, orABRAXANE®:Bevacizumab (VEGF targeting) complexes at the indicated dose.

FIG. 12 contains graphs plotting the percent BEV binding for ABX:BEVcomplexes exposed to 0.9% saline at room temperature or human plasma at37° C. for the indicated times.

FIG. 13 contains a line graph plotting the proliferation index for A375cells exposed to ABRAXANE® (ABX) only, cisplatin only, orABRAXANE®:cisplatin complexes at the indicated dose and contains a bargraph plotting demonstrating that 30% of cisplatin (CDDP) remainedunbound after ABX:cisplatin were mixed and incubated for 30 minutes.

FIG. 14 contains scatter plots of a flow cytometry analysis of theindicated complexes containing ABRAXANE®.

FIG. 15 contains photographs of Western blot analyses of the indicatedmaterials assessed for bevacizumab or taxol.

FIG. 16 contains graphs of the size distributions of the indicatedcomplexes incubated for the indicated time.

FIG. 17 contains graphs of the size distributions of the indicatedcomplexes incubated for one hour at room temperature.

FIG. 18 is a photograph of a Western blot analysis of ABX:BEV complexesexposed to serum for 15, 30, 45, or 60 minutes. The ABX:BEV complexeswere formed by incubating either 6 mg or 8 mg of BEV with ABX for 30minutes at room temperature. The primary antibody used for the Westernblot was an anti-paclitaxel antibody. Lane 1: ABX: BEV (6 mg) exposed toserum for 15 minutes; Lane 2: ABX: BEV (6 mg) exposed to serum for 30minutes; Lane 3: ABX: BEV (6 mg) exposed to serum for 45 minutes; Lane4: ABX: BEV (6 mg) exposed to serum for 60 minutes; Lane 5: blank; Lane6: ABX: BEV (8 mg) exposed to serum for 15 minutes; Lane 7: ABX: BEV (8mg) exposed to serum for 30 minutes; Lane 8: ABX: BEV (8 mg) exposed toserum for 45 minutes; Lane 9: ABX: BEV (8 mg) exposed to serum for 60minutes.

FIG. 19 is a photograph of a Western blot analysis of mixtures ofpaclitaxel (0.1, 0.5, 1, or 2 mg) and BEV (4 mg) incubated together for30 minutes at room temperature. The primary antibody used for theWestern blot was an anti-paclitaxel antibody. Lane 1: Bev (4 mg); Lane2: Taxol (2 mg); Lane 3: Taxol (2 mg)+Bev (4 mg); Lane 4: Taxol (1mg)+Bev (4 mg); Lane 5: Taxol (0.5 mg)+Bev (4 mg); Lane 6: Taxol (0.1mg)+Bev (4 mg).

FIG. 20 contains graphs plotting the particle size distribution forABX:BEV complexes as determined using a Mastersizer 2000E (MalvernInstruments Ltd., Worcestershire, England). ABX (20 mg/mL) and BEV (16,24, or 32 mg/mL) were incubated for 1, 2, or 4 hours at roomtemperature. After incubation, the mixtures were diluted 1:4 for a finalconcentration of ABX (5 mg/mL) and BEV (4, 6, or 8 mg/mL), and thediluted samples analyzed using a Mastersizer 2000E.

FIG. 21. After a 4 hour incubation at room temperature of ABX:BEVcomplexes (two different concentrations of ABX) in saline (left panel),soluble ABX/BEV complexes were detected by Western blot analysis thatmigrated in the MW range of approximately 200 kD. Identical bands wereidentified by Western blotting with anti-paclitaxel (α-paclitaxel) andanti-mouse IgG (α-mouse IgG) antibodies. Similarly, following incubationof ABX:BEV complexes in heparinized human plasma (right panel) at 37° C.for 15, 30, 45, or 60 minutes, the majority of the soluble paclitaxel(a-paclitaxel) migrated at a MW of 200 kD.

FIGS. 22A-C contain graphs plotting percent change (A), tumor size (B),and survival (C) for Group 1 mice treated with PBS, Bevacizumab (8mg/kg), ABRAXANE® (30 mg/kg), Bevacizumb (day 0, 8 mg/kg) followed byABRAXANE® (day 1, 30 mg/kg), or small nanoAB (complex).

FIGS. 23A-C contain graphs plotting percent change (A), tumor size (B),and survival (C) for Group 2 mice treated with PBS (day 0 and day7),Bevacizumab (8 mg/kg; day 0 and day7), ABRAXANE® (30 mg/kg; day 0 andday7), Bevacizumb (day 0, 8 mg/kg) followed by ABRAXANE® (day 1, 30mg/kg), or small nanoAB (complex; day 0 and day7).

FIGS. 24A-C contain graphs plotting percent change (A), tumor size (B),and survival (C) for Group 3 mice treated with PBS, Bevacizumab (24mg/kg), ABRAXANE® (30 mg/kg), Bevacizumb (day 0, 24 mg/kg) followed byABRAXANE® (day 1, 30 mg/kg), small nanoAB (nanoAB), or big nanoAB.

FIG. 25 is a graph plotting the particle size distribution for ABRAXANE®(ABX) dissolved in Bevacizumab (BEV) as determined using a Mastersizer2000E (Malvern Instruments Ltd., Worcestershire, England). ABX (10mg/mL) was reconstituted in 1 mL of the indicated amount of BEV, and themixtures were incubated at room temperature for 30 minutes.

FIG. 26 is a graph plotting the particle size distribution for ABRAXANE®(ABX) dissolved in Rituxan (RFT) as determined using a Mastersizer 2000E(Malvern Instruments Ltd., Worcestershire, England). ABX (10 mg/mL) wasreconstituted in 1 mL of the indicated amount of RIT, and the mixtureswere incubated at room temperature for 30 minutes.

FIG. 27 is a graph plotting the particle size distribution for ABRAXANE®(ABX) dissolved in Herceptin (HER) as determined using a Mastersizer2000E (Malvern Instruments Ltd., Worcestershire, England). ABX (10mg/mL) was reconstituted in 1 mL of the indicated amount of HER, and themixtures were incubated at room temperature for 30 minutes.

FIG. 28 is a graph plotting percent change at seven days in tumor sizefrom baseline of A375 tumor bearing nude mice treated with PBS,Bevacizumab (24 mg/kg) only, ABRAXANE®) (30 mg/kg) only, Bevacizumb (24mg/kg) followed the next day by

ABRAXANE® (30 mg/kg) (BEV+ABX), ABRAXANE® (30 mg/kg) followed the nextday by Bevacizumb (24 mg/kg) (ABX+BEV), and big nanoAB complexes (0.225μm; big nanoAB), in which ABRAXANE® (10 mg/mL) was premixed with 8 mg/kgBevacizumb and incubated for 30 minutes before injection.

FIG. 29 is a Kaplan Meier graph plotting survival of A375 tumor bearingnude mice treated with PBS, Bevacizumab (24 mg/kg) only, ABRAXANE® (30mg/kg) only, Bevacizumb (24 mg/kg) followed the next day by ABRAXANE®(30 mg/kg) (BEV+ABX), ABRAXANE® (30 mg/kg) followed the next day byBevacizumb (24 mg/kg) (ABX+BEV), and big nanoAB complexes (0.225 μm; bignanoAB), in which ABRAXANE® (10 mg/mL) was premixed with 8 mg/mLBevacizumb and incubated for 30 minutes before injection.

FIG. 30 is a graph plotting percent change at seven days in tumor sizefrom baseline of A375 tumor bearing nude mice treated intravenously withPBS, Bevacizumab (45 mg/kg) only, ABRAXANE® (30 mg/kg) only, Bevacizumb(45 mg/kg) followed the next day by ABRAXANE®) (30 mg/kg) (BEV+ABX),ABRAXANE® (30 mg/kg) followed the next day by Bevacizumb (45 mg/kg)(ABX+BEV), and nanoAB complexes of increasing sizes (0.16 μm, 0.225 μm,0.58 μm, and 1.13 μm).

FIG. 31 is a graph plotting tumor size of A375 tumors within nude micetreated intravenously with PBS, Bevacizumab (45 mg/kg) only, ABRAXANE®(30 mg/kg) only, Bevacizumb (45 mg/kg) followed the next day byABRAXANE® (30 mg/kg) (BEV+ABX), ABRAXANE® (30 mg/kg) followed the nextday by Bevacizumb (45 mg/kg) (ABX+BEV), and nanoAB complexes ofincreasing sizes (0.16 μm, 0.225 μm, 0.58 μm, and 1.13 μm).

FIG. 32 is a Kaplan Meier graph plotting survival of A375 tumor bearingnude mice treated intravenously with PBS, Bevacizumab (45 mg/kg) only,ABRAXANE® (30 mg/kg) only, Bevacizumb (45 mg/kg) followed the next dayby ABRAXANE® (30 mg/kg) (BEV+ABX), ABRAXANE® (30 mg/kg) followed thenext day by Bevacizumb (45 mg/kg) (ABX+BEV), and nanoAB complexes ofincreasing sizes (0.16 μm, 0.225 μm, 0.58 μm, and 1.13 μm).

FIG. 33 is a graph plotting the proliferation (proliferation index) ofA375 melanoma tumor cells treated in vitro with ABRAXANE® (ABX) 0-1000μg/mL, nanoAB (ABX:BEV) 0-1000 μg/mL, Cisplatin 0-200 μg/mL, or nanoABC(0-1000 μg/mL ABX, 4 mg/mL BEV, and 0-200 μg/mL Cisplatin).

FIG. 34 is a graph plotting the particle size distribution for particlesof ABRAXANE® (ABX) only, ABRAXANE® together with Bevacizumab (ABX:BEV),and ABRAXANE® together with Bevacizumab and Cisplatin (ABX:BEV:CIS; orABC) as determined using a Mastersizer 2000E (Malvern Instruments Ltd.,Worcestershire, England).

FIG. 35 is a graph plotting percent change at seven days in tumor sizefrom baseline of A375 tumor bearing nude mice treated intravenously withPBS, ABRAXANE® (30 mg/kg), cisplatin (2 mg/kg), nanoAB complexes (30mg/mL ABX and 8 mg/mL BEV), nanoAB (30 mg/mL ABX and 8 mg/mL BEV) pluscisplatin (2 mg/kg), and nanoABC (30 mg/kg ABX, 8 mg/kg BEV, and 2 mg/kgCis).

FIG. 36 is a Kaplan Meier graph plotting survival of A375 tumor bearingnude mice treated with PBS, ABRAXANE® (30 mg/kg), cisplatin (2 mg/kg),nanoAB complexes (30 mg/mL ABX and 8 mg/mL BEV), nanoAB (30 mg/mL ABXand 8 rng/mL BEV) plus cisplatin (2 mg/kg), and nanoABC (30 mg/kg ABX, 8mg/kg BEV, and 2 mg/kg Cis).

FIG. 37 is a graph plotting tumor size of A375 tumors within nude micetreated intravenously with PBS, ABRAXANE® (30 mg/kg), cisplatin (2mg/kg), nanoAB complexes (30 mg/mL ABX and 8 mg/mL BEV), nanoAB (30mg/mL ABX and 8 mg/mL BEV) plus cisplatin (2 mg/kg), and nanoABC (30mg/kg ABX, 8 mg/kg BEV, and 2 mg/kg Cis).

FIG. 38 is a set of flow cytometry results demonstrating the stabilityof nanoAB complexes incubated at room temperature for the indicateddurations.

FIG. 39 is a set of flow cytometry results demonstrating the stabilityof nanoAB complexes incubated at 37° C. in plasma for the indicateddurations.

FIG. 40 contains a photograph of a Western blot analysis of nanoABcomplexes incubated at 37° C. in plasma for the indicated durationsusing an anti-taxol antibody.

FIG. 41A contains fluorescent microscopy images and FIG. 41B containsscatter plots from a flow cytometry analysis. These studies, usingimmunofluorescent labeling of BEV and/or ABX, demonstrate dual labelingof the in vitro AB complexes, and suggest binding of BEV and ABX.

FIG. 42A contains a pie chart (top) demonstrating that 78% of thepaclitaxel content can be removed with centrifugation under conditionsused to remove particulate ABX. In the remaining supernatant, themajority (21%) of the paclitaxel is of a molecular weight >100kD,suggesting binding to BEV (140 kD), with a minor fraction (1%) of MWless than 100 kD. Western blot analysis (bottom) demonstrated that themajority of the non-particulate paclitaxel is of a molecular weight inthe 200 kD range, suggesting that free paclitaxel/albumin dimers (60 kD)may be binding to excess BEV (140 kD). Gel lanes: (1) ABX supernatant 45mg/mL after 4 h incubation at room temperature; (2) AB160 supernatant 1mg/mL, at 4 hours; (3) AB160 supernatant 10 mg/mL, at 4 hours; (4) ABXsupernatant overnight (45 mg/mL); (5) AB160 supernatant 1 mg/mL,overnight; (6) AB160 supernatant, 10 mg/mL, overnight.

FIG. 42B contains a cartoon (top) and an electron microscopy (EM) image(bottom) obtained by AB160 staining with anti-human-Ig gold conjugate,demonstrating that the median size of the AB160 complexes is in therange of 157 to 159 nm. This suggests a monolayer coating of the ABXnanoparticle by BEV.

FIG. 42C is a graph plotting cell counts from a flow-cytometry analysisof ABX and AB160 incubated with or without VEGF and an anti-VEGFfluorescinated monoclonal antibody, suggesting maximal VEGF binding tothe AB160 complex over that of ABX alone, and indicating that BEV bindsto the ABX albumin mantle via its Fc segment, preserving bindingaffinity for VEGF.

FIG. 42D is a picture of a non-denatured western blot, demonstrating theassociation of commercial human serum albumin (hSA, MW=60 kD) withbevacizumab (B, MW=140 kD).

FIGS. 43A and 43B contain a series of graphs plotting proliferation andVEGF binding for AB160 relative to either ABX or BEV, respectively.Under in vitro conditions, AB160 was equally as effective in inhibitinghuman melanoma proliferation (A375) as was seen with ABX alone (FIG.43A). In addition, AB160 was equally as efficient in binding solublehuman VEGF as was free BEV (FIG. 43B).

FIGS. 44A-44E are series of graphs plotting tumor volume in nude micebearing human A375 melanoma xenografts, in which tumors were allowed togrow to a size of 1000 mm³ before initiation of therapy. Mice receivedbevacizumab (BEV, FIG. 44A), nab-paclitaxel (ABX, FIG. 44B), bevacizumabfollowed by nab-paclitaxel (BEV+ABX, FIG. 44C), AB160 (FIG. 44D) orsaline (PBS, FIG. 44E). The absolute dose of nab-paclitaxel andbevacizumab was identical in all treatment cohorts. Each group includedat least 5 mice.

FIG. 44F is a graph plotting the % change in tumor size from baseline,on day 7 following treatment on day 0, for the groups of animals treatedas in FIGS. 44A-44E.

FIG. 44G is a Kaplan-Meier plot for the time from drug delivery until atumor size of 2500 mm³ was reached (pre euthanasia), as in FIGS.44A-44E.

FIGS. 45A-C contain graphs plotting data related to the in vivo biologicactivity of AB160. FIG. 45A is a graph plotting mouse plasma bevacizumablevels, demonstrating delayed plasma clearance of bevacizumab whenadministered as part of AB160 vs alone (five mice/cohort). FIG. 45B is agraph plotting the percentage of immunohistochemical staining forpaclitaxel of tumor tissues 24 hours after treatment with either saline(S), nab-paclitaxel (ABX) or AB160 (two mice per cohort, 5 random tissuesections), suggesting an approximately 50% increase in the number ofpaclitaxel positive staining tumor cells after mice were treated withAB160 over that of ABX alone. A similar increase in staining wasobserved with anti-human Ig IHC detecting human bevacizumab (greaternumber of cells in the AB160 cohort vs ABX or saline). FIG. 45C is agraph plotting the percentage of paclitaxel and Ig IHC staining for micetreated with ABX or AB160. There was no correlation (R values of 0.0229and 0.0176 per mouse) between the levels of paclitaxel and Ig IHCstaining for mice treated with ABX, but a suggestion of correlation (Rvalues of 0.7445 and 0.5496, per mouse) in mice treated with AB160.

DETAILED DESCRIPTION

This document provides methods and materials involved in treating cancer(e.g., skin cancers such as melanoma). For example, this documentprovides methods and materials for using complexes containingalbumin-containing nanoparticles (e.g., ABRAXANE® nanoparticles) andantibodies (e.g., anti-VEGF polypeptide antibodies such as AVASTIN®) totreat cancer.

The methods and materials provided herein can be used to treat any typeof cancer. For example, the methods and materials provided herein can beused to treat skin cancer (e.g., melanoma) and breast cancer. In somecases, the methods and materials provided herein can be used to treatcancer (e.g., skin cancer) in any type of mammal including, withoutlimitation, mice, rats, dogs, cats, horses, cows, pigs, monkeys, andhumans. When treating skin cancer, any type of skin cancer, such asmelanoma, can be treated using the methods and materials providedherein. For example, stage I, stage II, stage III, or stage IV melanomacan be treated. In some cases, a lymph node positive, a lymph nodenegative, or a metastatic melanoma can be treated as described herein.

In some cases, complexes containing albumin-containing nanoparticles(e.g., ABRAXANE® nanoparticles) and antibodies (e.g., anti-VEGFpolypeptide antibodies such as AVASTIN®) can be designed to have anaverage diameter that is greater than 1 μm. For example, appropriateconcentrations of albumin-containing nanoparticles and antibodies can beused such that complexes having an average diameter that is greater than1 μm are formed. In some cases, manipulations such as centrifugation canbe used to form preparations of albumin-containing nanoparticle/antibodycomplexes where the average diameter of those complexes is greater than1 μm. In some cases, the preparations of albumin-containingnanoparticle/antibody complexes provided herein can have an averagediameter that is between 1 μm and 5 μm (e.g., between 1.1 μm and 5 μm,between 1.5 μm and 5 μm, between 2 μm and 5 μμm, between 2.5 μm and 5μm, between 3 μm and 5 μm, between 3.5 μm and 5μm, between 4 μm and 5μm, between 4.5 μm and 5 μm, between 1.1 μm and 4.5 μm, between 1.1 μmand 4 μm, between 1.1 μm and 3.5 μm, between 1.1 μm and 3 μm, between1.1 μm and 2.5 μm, between 1.1 μm and 2 μm, or between 1.1 μm and 1.5μm). Preparations of albumin-containing nanoparticle/antibody complexesprovided herein having an average diameter that is between 1 μm and 5 μmcan be administered systemically (e.g., intravenously) to treat cancerslocated within a mammal's body. In some cases, the preparations ofalbumin-containing nanoparticle/antibody complexes provided herein canhave an average diameter that is between 5 μm and 50 μm (e.g., between 6μm and 50 μm, between 7 μm and 50 μm, between 10 μm and 50 μm, between15 μm and 50 μm, between 20 μm and 50 μm, between 25 μm and 50 μm,between 30 μm and 50 μm, between 35 μm and 50 pm, between 5 μm and 45μm, between 5 μm and 40 μm, between 5 μm and 35 μm, between 5 μm and 30pin, between 5 μm and 25 μm, between 5 μm and 20 μm, between 5 μm and 15μm, or between 10 μm and 30 μm). Preparations of albumin-containingnanoparticle/antibody complexes provided herein having an averagediameter that is between 5 μm and 50 μm can be administered into a tumor(e.g., intratumorally) or in a region of a tumor located within amammal's body.

In some cases, a preparation of albumin-containing nanoparticle/antibodycomplexes provided herein can have greater than 60 percent (e.g.,greater than 65, 70, 75, 80, 90, 95, or 99 percent) of the complexeshaving a diameter that is between 1 μm and 5 μm (e.g., between 1.1 μmand 5 μm, between 1.5 μm and 5 μm, between 2 μm and 5 μm, between 2.5 μmand 5 μm, between 3 μm and 5 μm, between 3.5 μm and 5 μm, between 4 μmand 5 μm, between 4.5 μm and 5 μm, between 1.1 μm and 4.5 μm, between1.1 μm and 4 μm, between 1.1 μm and 3.5 μm, between 1.1 μm and 3 μm,between 1.1 μm and 2.5 μm, between 1.1 μm and 2 μm, or between 1.1 μmand 1.5 μm). Preparation of albumin-containing nanoparticle/antibodycomplexes provided herein having greater than 60 percent (e.g., greaterthan 65, 70, 75, 80, 90, 95, or 99 percent) of the complexes with adiameter that is between 1 μm and 5 μm can be administered systemically(e.g., intravenously) to treat cancers located within a mammal's body.In some cases, a preparation of albumin-containing nanoparticle/antibodycomplexes provided herein can have greater than 60 percent (e.g.,greater than 65, 70, 75, 80, 90, 95, or 99 percent) of the complexeshaving a diameter that is between 5 μm and 50 μm (e.g., between 6 μm and50 μm, between 7 μm and 50 μm, between 10 μm and 50 μm, between 15 μmand 50 pm, between 20 μm and 50 μm, between 25 μm and 50 μm, between 30μm and 50 μm, between 35 μm and 50 μm, between 5 μm and 45 μm, between 5μm and 40 μm, between 5 μm and 35 μm, between 5 μm and 30 μm, between 5μm and 25 μm, between 5 μm and 20 μm, between 5 μm and 15 μm, or between10 μm and 30 μm). Preparation of albumin-containingnanoparticle/antibody complexes provided herein having greater than 60percent (e.g., greater than 65, 70, 75, 80, 90, 95, or 99 percent) ofthe complexes with a diameter that is between 5 μm and 50 μm can beadministered into a tumor (e.g., intratumorally) or in a region of atumor located within a mammal's body.

In some cases, complexes containing albumin-containing nanoparticles(e.g., ABRAXANE® nanoparticles) and antibodies (e.g., anti-VEGFpolypeptide antibodies such as AVASTIN®) can be designed to have anaverage diameter that is less than 1 μm.

For example, appropriate concentrations of albumin-containingnanoparticles and antibodies can be used such that complexes having anaverage diameter that is less than 1 pm are formed. In some cases, thepreparations of albumin-containing nanoparticle/antibody complexesprovided herein can have an average diameter that is between 0.1 μm and1 μm (e.g., between 0.1 μm and 0.95 μm, between 0.1 μm and 0.9 μm,between 0.1 μm and 0.8 μm, between 0.1 μm and 0.7 μm, between 0.1 μm and0.6 μm, between 0.1 μm and 0.5 μm, between 0.1 μmm and 0.4 μm, between0.1 μm and 0.3 μm, between 0.1 μm and 0.2 μm, between 0.2 82 m and 1 μm,between 0.3 μm and 1 μm, between 0.4 μm and 1 μm, between 0.5 μm and 1μm, between 0.2 μm and 0.6 μm, between 0.3 μm and 0.6 μm, between 0.2 μmand 0.5 μm, or between 0.3 μm and 0.5 μm). Preparations ofalbumin-containing nanoparticle/antibody complexes provided hereinhaving an average diameter that is between 0.1 μm and 0.9 μm can beadministered systemically (e.g., intravenously) to treat cancers locatedwithin a mammal's body.

In some cases, a preparation of albumin-containing nanoparticle/antibodycomplexes provided herein can have greater than 60 percent (e.g.,greater than 65, 70, 75, 80, 90, 95, or 99 percent) of the complexeshaving a diameter that is between 0.1 μm and 0.9 μm (e.g., between 0.1μm and 0.95 μm, between 0.1 μm and 0.9 μm, between 0.1 μm and 0.8 μm,between 0.1 μm and 0.7 μm, between 0.1 μm and 0.6 μm, between 0.1 μm and0.5 μm, between 0.1 μm and 0.4 μm, between 0.1 μm and 0.3 μm, between0.1 μm and 0.2 μm, between 0.2 μm and 1 μm, between 0.3 μm and 1μm,between 0.4 μm and 1 pm, between 0.5 μm and 1 μm, between 0.2 μm and 0.6μm, between 0.3 μm and 0.6 μm, between 0.2 μm and 0.5 μm, or between 0.3μm and 0.5 μm). Preparation of albumin-containing nanoparticle/antibodycomplexes provided herein having greater than 60 percent (e.g., greaterthan 65, 70, 75, 80, 90, 95, or 99 percent) of the complexes with adiameter that is between 0.1 μm and 0.9 μm can be administeredsystemically (e.g., intravenously) to treat cancers located within amammal's body.

In general, albumin-containing nanoparticles such as ABRAXANE® can becontacted with an antibody such as an anti-VEGF polypeptide antibody(e.g., AVASTIN®) prior to administration to a human to form analbumin-containing nanoparticle/antibody complex (e.g., anABRAXANE®/anti-VEGF polypeptide antibody complex). Any appropriatealbumin-containing nanoparticle preparation and any appropriate antibodycan be used as described herein. For example, ABRAXANE® nanoparticlescan be used as described herein. Examples of antibodies that can be usedto form albumin-containing nanoparticle/antibody complexes as describedherein include, without limitation, bevacizumab (AVASTIN®), trastuzamab,and rituxan. For example, an appropriate dose of ABRAXANE® and anappropriate dose of AVASTIN® can be mixed together in the samecontainer. This mixture can be incubated at an appropriate temperature(e.g., room temperature, between 15 ° C. and 30 ° C., between 15 ° C.and 25 ° C., between 20 ° C. and 30 ° C., or between 20 ° C. and 25 °C.) for a period of time (e.g., about 30 minutes, or between about 5minutes and about 60 minutes, between about 5 minutes and about 45minutes, between about 15 minutes and about 60 minutes, between about 15minutes and about 45 minutes, between about 20 minutes and about 400minutes, or between about 25 minutes and about 35 minutes) before beingadministered to a cancer patient (e.g., a melanoma patient). In somecases, ABRAXANE can be contacted with an anti-VEGF polypeptide antibodyby injecting both ABRAXANE® and the anti-VEGF polypeptide antibodyeither individually or as a pre-mixed combination into an IV bagcontaining an IV bag solution. The contents of the IV bag includingABRAXANE®/anti-VEGF polypeptide antibody complexes can be introducedinto the patient to be treated. In some cases, albumin-containingnanoparticles such as ABRAXANE® can be contacted with an antibody suchas an anti-VEGF polypeptide antibody (e.g., AVASTIN®) to formalbumin-containing nanoparticle/antibody complexes (e.g.,ABRAXANE®/anti-VEGF polypeptide antibody complexes) that are storedprior to being administered to a cancer patient (e.g., a melanomapatient). For example, a composition containing albumin-containingnanoparticle/antibody complexes can be formed as described herein andstored for a period of time (e.g., days or weeks) prior to beingadministered to a cancer patient.

Any appropriate method can be used to obtain albumin-containingnanoparticles such as ABRAXANE® and an antibody such as an anti-VEGFpolypeptide antibody. For example, ABRAXANE® can be obtained fromCelgene Corp. or as described elsewhere (U.S. Pat. No. 6,537,579).AVASTIN® can be obtained from Genentech Corp. or Roche Corp. or asdescribed elsewhere (U.S. Pat. No. 6,054,297).

In some cases, the combination of an albumin-containing nanoparticlesuch as ABRAXANE® and an antibody such as anti-VEGF polypeptide antibodycan include one or more other agents such as an alkylating agent (e.g.,a platinum compound). Examples of platinum compounds that can be used asan alkylating agent include, without limitation, carboplatin(PARAPLATIN®), cisplatin (PLATINOL®), oxaliplatin (ELOXATIN®), andBBR3464. Examples of other agents that can be included within analbumin-containing nanoparticle/antibody complex provided hereininclude, without limitation, bendamustine, bortezomib, cabazitaxel,chlorambucil, dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib,etoposide, everolimus, gefitinib, idarubicin, hydroxyurea, imatinib,lapatinib, melphalan, mitoxantrone, nilotinib, oxaliplatin, pazopanib,pemetrexed, romidepsin, sorafenib, sunitinib, teniposide, vinblastine,and vinorelbine.

Any appropriate method can be used to administer an albumin-containingnanoparticle/antibody complex provided herein (e.g.,ABRAXANE®)/anti-VEGF polypeptide antibody complexes) to a mammal. Forexample, a composition containing albumin-containingnanoparticle/antibody complexes such as ABRAXANE®/anti-VEGF polypeptideantibody complexes can be administered via injection (e.g., subcutaneousinjection, intramuscular injection, intravenous injection, orintrathecal injection).

Before administering a composition containing an albumin-containingnanoparticle/antibody complex provided herein (e.g., ABRAXANE®/anti-VEGFpolypeptide antibody complexes) to a mammal, the mammal can be assessedto determine whether or not the mammal has cancer (e.g., skin cancer).Any appropriate method can be used to determine whether or not a mammalhas cancer (e.g., skin cancer). For example, a mammal (e.g., human) canbe identified as having skin cancer using standard diagnostictechniques. In some cases, a tissue biopsy can be collected and analyzedto determine whether or not a mammal has skin cancer.

After identifying a mammal as having cancer (e.g., skin cancer), themammal can be administered a composition containing albumin-containingnanoparticle/antibody complexes provided herein (e.g.,ABRAXANE®/anti-VEGF polypeptide antibody complexes). For example, acomposition containing ABRAXANE®)/anti-VEGF polypeptide antibodycomplexes can be administered prior to or in lieu of surgical resectionof a tumor. In some cases, a composition containing albumin-containingnanoparticle/antibody complexes provided herein (e.g.,ABRAXANE®/anti-VEGF polypeptide antibody complexes) can be administeredfollowing resection of a tumor.

A composition containing albumin-containing nanoparticle/antibodycomplexes provided herein (e.g., ABRAXANE®/anti-VEGF polypeptideantibody complexes) can be administered to a mammal in any appropriateamount, at any appropriate frequency, and for any appropriate durationeffective to achieve a desired outcome (e.g., to increaseprogression-free survival). In some cases, a composition containingalbumin-containing nanoparticle/antibody complexes provided herein(e.g., ABRAXANE®/anti-VEGF polypeptide antibody complexes) can beadministered to a mammal having cancer (e.g., skin cancer) to reduce theprogression rate of the cancer (e.g., melanoma) by 5, 10, 25, 50, 75,100, or more percent. For example, the progression rate can be reducedsuch that no additional cancer progression is detected. Any appropriatemethod can be used to determine whether or not the progression rate ofcancer (e.g., skin cancer) is reduced.

For example, the progression rate of skin cancer can be assessed byimaging tissue at different time points and determining the amount ofcancer cells present. The amounts of cancer cells determined withintissue at different times can be compared to determine the progressionrate. After treatment as described herein, the progression rate can bedetermined again over another time interval. In some cases, the stage ofcancer (e.g., skin cancer) after treatment can be determined andcompared to the stage before treatment to determine whether or not theprogression rate was reduced.

In some cases, a composition containing albumin-containingnanoparticle/antibody complexes provided herein (e.g.,ABRAXANE®/anti-VEGF polypeptide antibody complexes) can be administeredto a mammal having cancer (e.g., skin cancer) under conditions whereprogression-free survival is increased (e.g., by 5, 10, 25, 50, 75, 100,or more percent) as compared to the median progression-free survival ofcorresponding mammals having untreated cancer (e.g., untreated skincancer) or the median progression-free survival of corresponding mammalshaving cancer (e.g., skin cancer) treated with ABRAXANE® and an antibody(e.g., an anti-VEGF polypeptide antibody) without formingABRAXANE®/antibody complexes (e.g., without forming ABRAXANE®/anti-VEGFpolypeptide antibody complexes). In some cases, a composition containingalbumin-containing nanoparticle/antibody complexes provided herein(e.g., ABRAXANE®/anti-VEGF polypeptide antibody complexes) can beadministered to a mammal having cancer (e.g., skin cancer) to increaseprogression-free survival by 5, 10, 25, 50, 75, 100, or more percent ascompared to the median progression-free survival of correspondingmammals having cancer (e.g., skin cancer) and having received ABRAXANE®or an antibody (e.g., an anti-VEGF polypeptide antibody) alone.Progression-free survival can be measured over any length of time (e.g.,one month, two months, three months, four months, five months, sixmonths, or longer).

In some cases, a composition containing albumin-containingnanoparticle/antibody complexes provided herein (e.g.,ABRAXANE®/anti-VEGF polypeptide antibody complexes) can be administeredto a mammal having cancer (e.g., skin cancer) under conditions where the8-week progression-free survival rate for a population of mammals is 65%or greater (e.g., 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80% or greater) than that observed in a population ofcomparable mammals not receiving a composition containingalbumin-containing nanoparticle/antibody complexes provided herein(e.g., ABRAXANE®/anti-VEGF polypeptide antibody complexes). In somecases, a composition containing albumin-containing nanoparticle/antibodycomplexes provided herein (e.g., ABRAXANE®/anti-VEGF polypeptideantibody complexes) can be administered to a mammal having cancer (e.g.,skin cancer) under conditions where the median time to progression for apopulation of mammals is at least 150 days (e.g., at least 155, 160,163, 165, or 170 days).

An effective amount of a composition containing albumin-containingnanoparticle/antibody complexes provided herein (e.g.,ABRAXANE®/anti-VEGF polypeptide antibody complexes) can be any amountthat reduces the progression rate of cancer (e.g., skin cancer),increases the progression-free survival rate, or increases the mediantime to progression without producing significant toxicity to themammal. Typically, an effective amount of ABRAXANE® can be from about 50mg/m² to about 150 mg/m² (e.g., about 80 mg/m²), and an effective amountof an anti-VEGF polypeptide antibody such as bevacizumab can be fromabout 5 mg/kg to about 20 mg/kg (e.g., about 10 mg/kg). If a particularmammal fails to respond to a particular amount, then the amount ofABRAXANE® or anti-VEGF polypeptide antibody can be increased by, forexample, two fold. After receiving this higher concentration, the mammalcan be monitored for both responsiveness to the treatment and toxicitysymptoms, and adjustments made accordingly. The effective amount canremain constant or can be adjusted as a sliding scale or variable dosedepending on the mammal's response to treatment. Various factors caninfluence the actual effective amount used for a particular application.For example, the frequency of administration, duration of treatment, useof multiple treatment agents, route of administration, and severity ofthe cancer (e.g., skin cancer) may require an increase or decrease inthe actual effective amount administered.

The frequency of administration can be any frequency that reduces theprogression rate of cancer (e.g., skin cancer), increases theprogression-free survival rate, or increases the median time toprogression without producing significant toxicity to the mammal. Forexample, the frequency of administration can be from about once a monthto about three times a month, or from about twice a month to about sixtimes a month, or from about once every two months to about three timesevery two months. The frequency of administration can remain constant orcan be variable during the duration of treatment. A course of treatmentwith a composition containing ABRAXANE®/anti-VEGF polypeptide antibodycomplexes can include rest periods. For example, a compositioncontaining ABRAXANE®/anti-VEGF polypeptide antibody complexes can beadministered over a two week period followed by a two week rest period,and such a regimen can be repeated multiple times. As with the effectiveamount, various factors can influence the actual frequency ofadministration used for a particular application. For example, theeffective amount, duration of treatment, use of multiple treatmentagents, route of administration, and severity of the skin cancer mayrequire an increase or decrease in administration frequency.

An effective duration for administering a composition provided hereincan be any duration that reduces the progression rate of cancer (e.g.,skin cancer), increases the progression-free survival rate, or increasesthe median time to progression without producing significant toxicity tothe mammal. Thus, the effective duration can vary from several days toseveral weeks, months, or years. In general, the effective duration forthe treatment of skin cancer can range in duration from several weeks toseveral months. In some cases, an effective duration can be for as longas an individual mammal is alive. Multiple factors can influence theactual effective duration used for a particular treatment. For example,an effective duration can vary with the frequency of administration,effective amount, use of multiple treatment agents, route ofadministration, and severity of the cancer (e.g., skin cancer).

A composition containing albumin-containing nanoparticle/antibodycomplexes provided herein (e.g., ABRAXANE®/anti-VEGF polypeptideantibody complexes) can be in any appropriate form. For example, acomposition provided herein can be in the form of a solution or powderwith or without a diluent to make an injectable suspension. Acomposition also can contain additional ingredients including, withoutlimitation, pharmaceutically acceptable vehicles. A pharmaceuticallyacceptable vehicle can be, for example, saline, water, lactic acid,mannitol, or combinations thereof.

After administering a composition provided herein to a mammal, themammal can be monitored to determine whether or not the cancer (e.g.,skin cancer) was treated. For example, a mammal can be assessed aftertreatment to determine whether or not the progression rate of melanomawas reduced (e.g., stopped). As described herein, any method can be usedto assess progression and survival rates.

In some cases, a formulation of ABRAXANE®/AVASTIN® complexes describedin Example 1 can be administered to a human melanoma patient asdescribed in the methods set forth in Example 10.

In some cases, nanoparticles containing albumin (e.g., nanoparticleswith an albumin shell) and an agent other than placitaxel can be used asdescribed herein in place of or in combination with ABRAXANE®. Forexample, albumin-containing nanoparticles designed to carry a cancerchemotherapeutic agent can be used to form nanoparticle/anti-VEGFpolypeptide antibody complexes that can be used as described herein. Anexample of such a cancer chemotherapeutic agent includes, withoutlimitation, vinblastine.

In some cases, a composition can be formulated to include nanoparticlescontaining albumin (e.g., nanoparticles with an albumin shell) that areconjugated to an antibody, agent, or combination of antibodies andagents listed in Table 1 to form complexes for treating cancer. Forexample, albumin nanoparticles can be formulated to include Cetuximab totreat head and neck cancer. In some cases, albumin nanoparticles can beformulated to include Cetuximab and vinblastine as complexes to treathead and neck cancer. In some cases, a composition can be formulated toinclude nanoparticles containing albumin (e.g., nanoparticles with analbumin shell) that are conjugated to a combination of differentantibodies or agents listed in Table 1 to form complexes capable oftreating multiple different cancers. For example, albumin nanoparticlescan be formulated to include Herceptin, Bevacizurmab, and Docetaxel ascomplexes for treating breast cancer and ovarian cancer.

TABLE 1 List of possible antibodies and agents for forming anti-cancercomplexes with albumin. Cancer Antibody Agent Head and neck cancerCetuximab vinblastine Breast cancer Herceptin Docetaxel; doxorubicin;epirubicin; Everolimus; gefitinib; lapatinib; mitoxantrone; pemetrexed;sunitinib; vinblastine; vinorelbine Colon cancer Bevacizumab;Oxaliplatin; pemetrexed; Cetuximab; sunitinib Panitumumab Ovarian cancerBevacizumab Docetaxel; doxorubicin; epirubicin; hydroxyurea; melphalan;oxaliplatin; pazopanib Lung cancer Bevacizumab Docetaxel; doxorubicin;epirubicin; erlotinib; etoposide; gefitinib; pazopanib; pemetrexed;sunitinib; vinblastine; vinorelbine Pancreatic cancer Erlotinib;sunitinib Bladder cancer Doxorubicin; pemetrexed myeloma Bortezomib;melphalan CLL/lymphoma Ofatumumab; Bendamustine; Alemtuzumab Prostatecancer Cabazitaxel; docetaxel CLL chlorambucil CML/ALL dasatinib Stomachcancer Herceptin Doxorubicin; epirubicin Leukemia (AML, ANLL, RituximabDoxorubicin; idarubicin; ALL) imatinib; mitoxantrone; nilotinib;teniposide Hodgkin's disease Chlorambucil; doxorubicin; vinblastinenon-Hodgkin's lymphoma Chlorambucil; doxorubicin; mitoxantrone Thyroidcancer Doxorubicin Bone sarcoma Doxorubicin Wilms' tumor DoxorubicinKaposi's sarcoma Etoposide Ewing's sarcoma Etoposide Testicular cancerEtoposide; vinblastine Lymphoma Rituximab Etoposide; romidepsin renalcell carcinoma Bevacizumab Everolimus; pazopanib; sorafenib; sunitinibmelanoma Hydroxyurea; melphalan gastrointestinal stromal Imatinib;sunitinib tumors Soft tissue sarcoma pazopanib Cervical cancerpemetrexed Hepatocellular carcinoma sorafenib

In some cases, nanoparticles containing albumin (e.g., nanoparticleswith an albumin shell) or a complex described herein (e.g.,ABRAXANE®/AVASTIN® complexes) can be formulated to include one or moreanti-chronic inflammation treatment agents designed to reduce the globalstate of immune dysfunction and/or chronic inflammation present within acancer patient. For example, steroidal anti-inflammatory agents (e.g.,prednisone), non-steroidal anti-inflammatory agents (e.g., naproxen),lympho-depleting cytotoxic agents (e.g., cyclophosphamide), immune celland/or cytokine targeting antibodies (e.g., infliximab), or acombination thereof can be incorporated into nanoparticles containingalbumin or ABRAXANE®/AVASTIN® complexes. In some cases, anti-IL-4 agents(e.g., anti-IL-4 antibodies), anti-IL-13 agents (e.g., soluble IL-13receptor), and combinations thereof can be incorporated intonanoparticles containing albumin or ABRAXANE®/AVASTIN® complexes.

Any appropriate method can be used to assess whether or not the globalstate of immune dysfunction and/or chronic inflammation was reducedfollowing an anti-chronic inflammation treatment. For example, cytokineprofiles (e.g., IL-4, IL-13, IL-4, IL-13, IL-5, IL-10, IL-2, andinterferon gamma) present in blood can be assessed before and after ananti-chronic inflammation treatment to determine whether or not theglobal state of immune dysfunction and/or chronic inflammation wasreduced.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1

Contacting ABRAXANE® with AVASTIN® results in the formation ofABRAXANE®/AVASTIN® complexes ABRAXANE® (1 mg/mL) and AVASTIN (25 mg/mL)were stored at 4° C. 10 μg (10 μL) of ABRAXANE® nanoparticles and 500 μg(20 μL) of AVASTIN were mixed in a total volume of 30 μL. The ABRAXANE®and AVASTIN were incubated at room temperature for 30 minutes.

After incubation, the ABRAXANE® nanoparticles were spun and washed threetimes with 1× PBS to eliminate unbound bevacizumab. The nanoparticleswere spun at 5000 rpm for 5 minutes and resuspended in 50 μL of 1× PBS.

100 ng or 500 ng of VEGF was added to each tube for 30 minutes at roomtemperature, and the washes were repeated to eliminate unbound VEGF. PEanti-human VEGF was added at a 1:50 dilution, and the particles wereonce again incubated and washed. Visualization was done by flowcytometry, and percentage of PE (VEGF) positive particles was determined(FIGS. 1-4). Various combinations of agents were tested as indicated inthe figures. These results demonstrate that ABRAXANE® and bevacizumabspontaneously associate in a manner that preserves VEGF bindingpotential.

ABRAXANE® nanoparticles were mixed with varying concentrations ofbevacizumab (0.5, 5, 10, and 25 mg/mL). The particles were viewed bylight microscopy at 4 and 24 hours after mixing. The macromolecular sizeof the ABX:BEV complexes was dependent on the concentration of thebevacizumab added and the ABRAXANE® nanoparticles (FIG. 9). Once amaximum size was reached, the ABX:BEV complexes began to break downwithin about 24 hours (FIG. 9).

Bevacizumab was added to ABRAXANE® nanoparticles in varyingconcentrations (0.5, 5, 10, 25 mg/mL) and incubated for 30 minutes atroom temperature to allow complex formation. ABRAXANE® nanoparticlesalone, ABX:BEV complexes, and 2 μm standard beads were visualized byflow cytometry. The complex size increased with increased concentrationsof bevacizumab (FIG. 10). The larger the particle-size, the further tothe right the peak will be. These results demonstrate that complex sizecan be manipulated by varying the concentration of bevacizumab added.

In another study, ABRAXANE® nanoparticles and bevacizumab were incubatedtogether for 4 hours and overnight at 1 mg/mL or 10 mg/mL. ABRAXANE®nanoparticles alone were also incubated for 4 hours and overnight as acontrol. After the allotted time was reached, the complexes were spundown at 7500 RPM for 5 minutes. The supernatants were collected andmixed 1:1 with Laemmli buffer and boiled at 100 degrees for 3 minutes.20 μL of sample was loaded onto a 7.5% Tris-HCl Criteron gel.

A high range molecular weight marker (BioRad) was added for sizedetermination. The gel was run for 3 hours at 75V.

After the gel ran to completion, the gel was placed in a transfercassette so the proteins could be moved onto a PVDF membrane. Thetransfer took place overnight at 4° C. running at 20V. The membrane wasremoved and rocked in TBST containing 5% milk to block for 3 hours atroom temperature. The primary antibodies used were Rabbit anti-Taxol(1:500 dilution) and goat anti-mouse IgG-Fab specific-IIRP conjugated(1:500 dilution). Antibodies were diluted into 10 mL of TBST with 5%milk. Primary antibodies were allowed to bind overnight at 4° C. whilerocking.

Primary antibodies were removed, and the membranes were washed threetimes for 10 minutes with TBST. The taxol blot was incubated in a 1:1000dilution of secondary anti-rabbit IgG-HRP for 1.5 hours rocking at roomtemperature. The anti-mouse IgG (Bevacizumab) membrane was incubated inECL detection reagent (GE Amershem) for 5 minutes before it was exposedto film. Membrane was exposed for 10 seconds, 1 minute, and 5 minutes.

After the incubation in secondary antibody, the taxol blot was washedwith TBST for 10 minutes three times. The membrane was then placed inECL detection reagent for 5 minutes and exposed to film. The exposuretimes were 1 second, 2 seconds, and 10 seconds.

The IgG blot was specific for the mouse portion of the bevacizumabhumanized antibody. A clear concentration dependent increase fromcomplexes mixed at 1 mg/mL to 10 mg/mL was observed (FIG. 15). Taxol isa small molecule around 20 kDa. Free taxol was observed at the bottom ofthe blot, but it also was observed running at the bevacizumab molecularweight (149 kDa; FIG. 15). These results demonstrate that taxol wasbound to the bevacizumab in the supernatant after the large particleswere removed by centrifugation.

In another study, ABRAXANE® nanoparticles and bevacizumab were incubatedfor various times (1, 4, and 12 hours), and the particle sizedistribution of the resulting complexes was determined relative toABRAXANE®nanoparticles alone using the Malvern Mastersizer 2000E. Thesize of the complexes generated was a function of antibody concentrationand incubation time (FIGS. 16 and 17). In FIGS. 16, 1 and 10 mg/mL ofbevacizumab was incubated with ABRAXANE nanoparticles for 4 hours andovernight. The complexes generated with 10 mg/mL bevacizumab were muchlarger (8.479 μm) than those with 1 mg/mL bevacizumab (0.165 μm). Afteran overnight incubation, the larger complexes began to break down.

In FIG. 17, complex size increased with concentration of bevacizumabadded when incubated for 1 hour at room temperature. In addition, largercomplexes were formed when 1 mg/mL bevacizumab was incubated withABRAXANE® nanoparticles, spun, and resuspended as compared to the sizeobserved when the same amount (1 mg/mL) of bevacizumab was incubatedwith ABRAXANE® nanoparticles without spinning the preparation (FIG. 17).These results demonstrate that complex size can be manipulated byaltering concentrations, by manual forces (e.g., centrifugation), or byboth.

In another study, ABRAXANE®nanoparticles were dissolved at aconcentration of 20 mg/mL, and bevacizumab was added at a finalconcentration of 16, 24, or 32 mg/mL. The mixtures were incubated atroom temperature for various times (1, 2, and 4 hours). After thisincubation, the mixture was diluted 1:4 (final concentration ofABRAXANE=5 mg/mL; final concentrations of bevacizumab=4, 6, or 8 mg/mL).The particle size distribution of the resulting complexes was determinedrelative to ABRAXANE® nanoparticles alone using the Malvern Mastersizer2000E. The size of the complexes generated was a function of antibodyconcentration and incubation time (FIG. 20).

In another study, 10 mg of ABRAXANE® nanoparticles was reconstituted in1 mL of bevacizumab at 0, 2, 4, 6, 8, 10, 15, or 25 mg/mL, and themixture was incubated for 1 hour at room temperature. The particle sizedistribution of the resulting complexes was determined bylight-refraction of unlabeled complexes (Table 2). The size of thecomplexes generated was a function of antibody concentration (Table 2).

TABLE 2 ABX BEV d (0.1) d (0.5) d (0.9) (mg/mL) (mg/mL) μm μm μm 10 00.125 0.146 0.174 10 2 0.122 0.157 0.196 10 4 0.138 0.159 0.182 10 60.124 0.174 0.235 10 8 0.171 0.226 0.278 10 10 0.516 0.577 0.67 10 150.981 1.129 1.31 10 25 1.036 2.166 3.233

ABRAXANE and bevacizmab were mixed and incubated for 30 minutes at roomtemperature to allow complex formation. Mice were injected with 100 μLof the complexes containing 5 mg of ABRAXANE and 1 mg of bevacizumab inthe dorsal tail vein. Injection of the complexes did not harm any mice.

Example 2

Human Plasma Inhibits the Formation of ABRAXANE®/AVASTIN® complexes

10 μL (10 μg) of ABRAXANE® was added to eppendorf tubes, and 500 μg (25μL) of AVASTIN was added and resuspended in a final volume of 50 μL.Human plasma was titrated using 1:2 dilutions (1:2, 1:4, 1:8, or 1:16).50 μL of plasma and 50 μL of each plasma titration were added to thetubes with ABRAXANE® and AVASTIN. In some cases, human serum albumin(500 μg, 50 μg, 5 μg, 0.5 _(μg, or) 0.05 μg/mL) or human polyclonalimmunoglobulin (500 μg, 50 μg, 5 μg, 0.5 μg, and 0.05 μg/mL) was addedto the tubes in place of human plasma.

After a 30 minute incubation at room temperature, the ABRAXANE®nanoparticles were washed in 1× PBS twice. 100 ng of VEGF was added toeach tube for 30 minutes at room temperature, and the washes wererepeated. PE anti-human VEGF was added at a 1:50 dilution, and particleswere once again incubated and washed. Visualization was done by flowcytometry, and percentage of PE (VEGF) positive particles was determined(FIG. 5-8).

Example 3

ABRAXANE®/AVASTIN® Complexes have a Higher Level of Cell Toxicity thanABRAXANE® Alone or ABRAXANE®/Herceptin Complexes

The VEGF producing melanoma tumor cell line, A375, was incubatedovernight in the presence of ABRAXANE®nanoparticles only, ABRAXANE®Herceptin (non-VEGF targeting) complexes, and ABRAXANE®/AVASTIN®(ABX:BEV; VEGF targeting) complexes. Increasing doses of drug were addedto the cells to give 6.25, 12.5, 25, 50, 100, and 200 μg/mL of taxol.After the overnight incubation, cell proliferation was determined bymeasuring the level of DNA synthesis. A higher level of cell toxicity(less DNA synthesis) of cells incubated with the VEGF targetingcomplexes (ABX:BEV) relative the ABX alone and non-VEGF targetedcomplexes (ABX:HER) (FIG. 11).

Example 4 Stability of ABRAXANE®/AVASTIN® Complexes

ABRAXANE®/AVASTIN® complexes were fluorescently labeled such that boththe albumin of the ABRAXANE® and the bevacizumab were directly labeledwith a fluorescent marker. The complexes were visualized by flowcytometry after 0, 1, 2, 3, 4, 24, and 48 hours in 0.9% saline at roomtemperature and after 0, 15, 30, 60, and 120 minutes in human plasma at37° C. The complexes were stable in saline at room temperature with onlyabout 10% loss at 24 hours (FIG. 12). In human plasma at 37° C., thecomplexes began to break down in about 15 minutes and were completelyundetectable by 120 minutes.

Example 5 ABRAXANE®/Cisplatin Complexes

ABRAXANE® nanoparticles were incubated with cisplatin (cisplatinum orcis-diamminedichloroplatinum(II) (CDDP)) for 30 minutes at 37° C. Theparticles were spun, and the supernatant was tested by HPLC to determinehow much free cisplatin was in suspension. Cisplatin spontaneously boundto the ABRAXANE® nanoparticles, and the amount remaining in suspensionafter the 30 minute incubation with the ABRAXANE® nanoparticles was onlyabout 30% of the original concentration (FIG. 13). These resultsdemonstrate that about 70% of the cisplatin bound to the ABRAXANE®nanoparticles.

In another experiment, ABRAXANE®/cisplatin complexes were generated asdescribed above and added to A375 tumor cells. After an overnightincubation, proliferation of the cells was measured by determining thelevel of DNA synthesis. The toxicity of the ABRAXANE®/cisplatincomplexes was measured relative to the two drugs individually. TheABRAXANE®/cisplatin complexes were more toxic to cells (lower level ofDNA synthesis) than ABRAXANE® alone but less toxic than cisplatin alone(FIG. 13). These results demonstrate that cisplatin can be bound toABRAXANE® nanoparticles and delivered to tumors without the highly toxicside effects of cisplatin alone.

Example 6 ABRAXANE®/Antibody Complexes

Three therapeutic monoclonal antibodies (bevacizumab, trastuzamab, andrituxan) were fluorescently labeled and incubated with fluorescentlylabeled ABRAXANE® nanoparticles. The particles were spun down, washed,and visualized by flow cytometry. All three of these recombinanttherapeutic antibodies spontaneously formed complexes with ABRAXANE®nanoparticles (FIG. 14). These results demonstrate thatalbumin-containing nanoparticles can be used to form larger complexesnot only with bevacizumab antibodies but also with other antibodies suchas trastuzamab and rituxan.

Taken together, the results provided herein demonstrate that in vitromixing of albumin-containing nanoparticles (e.g., ABRAXANE®nanoparticles) and antibodies (e.g., bevacizumab, trastuzamab, orrituxan) leads to macromolecular complex formation, the characteristicsof which (e.g., size, antibody content, or chemotherapeutic drugcontent) can be customized depending on need. These results alsodemonstrate that the macromolecular complexes retain antibody mediatedtarget binding specificity, retain or exhibit enhanced chemotherapeutictumor cell cytotoxicity, and exhibit no additional toxicity beyond thatof ABRAXANE® nanoparticles alone.

Example 7

ABRAXANE®/AVASTIN® complexes disassociate in serum The following wasperformed to determine what happens to ABRAXANE®/AVASTIN® complexes inserum over time. 6 mg or 8 mg of AVASTIN® were bound to ABRAXANE® for 30minutes at room temperature. The complexes were incubated with serum for15, 30, 45, or 60 minutes. After this incubation, the serum/complexsolution was spun down at 10,000 rpm for 10 minutes at 4° C. Thesupernatants were collected, separated using gel electrophoresis, andanalyzed via Western blotting with an anti-paclitaxel antibody and anHRP-conjugated secondary antibody.

Incubation in the presence of serum resulted in complex disassociation,not disintegration (FIG. 18).

In another experiment, ABRAXANE®/AVASTIN® complexes (in saline orplasma) appeared to dissociate primarily into ABX/BEV complexes (FIG.21). Results appeared to suggest that the ABRAXANE®/AVASTIN® associationis mediated via albumin binding to the Fc fragment of AVASTIN®.

Example 8 Bevacizumab Does not Bind Free Paclitaxel

The following was performed to determine if bevacizumab binds freepaclitaxel. 4 mg of bevacizumab was incubated with paclitaxel (0.1, 0.5,1, or 2 mg) for 30 minutes at room temperature. After this incubation,the mixtures were separated using gel electrophoresis and analyzed viaWestern blotting with an anti-paclitaxel antibody and an HRP-conjugatedsecondary antibody.

Bevacizumab did not bind free paclitaxel (FIG. 19).

Example 9

ABRAXANE®/AVASTIN® Complexes Inhibit Tumor Growth More Effectively thanABRAXANE® alone, AVASTIN® alone, and the Sequential Use of ABRAXANE® andAVASTIN®

Female athymic nude mice were injected with 1×10⁶ A375 melanoma cells.Tumors were allowed to grow, and treatments were administered whentumors were between 600 and 1000 mm³. Mice were treated with (a) 100 μLPBS, (b) Bevacizumab (8 mg/kg for Group I and II; 24 mg/kg for GroupIII), (c) ABRAXANE® (30 mg/kg), (d) Bevacizumb (day 0, 8 mg/kg for GroupI and II; 24 mg/kg for Group III) followed by ABRAXANE® (day 1, 30mg/kg), (e) small nanoAB (Group I, II, and III), or (f) big nanoAB(Group III). Mice were treated one time for Groups I and III and twotimes (day 0 and day 7) for Group II. Tumor size was monitored 2-3 timesper week. Mice were sacrificed when tumors reached 2000-2500 mm³.Percent change from baseline was calculated by [(tumor size on day 7(Group I and III) or day 21 (Group II)—tumor size on day oftreatment)/tumor size on day of treatment]*100.

Small nanoAB (also nanoAB or Complex in Group I and II when only onesize nanoparticle was tested) was produced as follows. 10 mg ABRAXANE®was reconstituted in 3.6 mg of Bevacizumab in 500 μL 0.9% saline andincubated for 1 hour at room temperature. After incubation, nanoAB wasbrought to 1 mL with 0.9% saline. NanoAB was further diluted, and 100 μLwas administered to mice for an 8 mg/kg bevacizumab and 30 mg/kgABRAXANE® dose. Average particle size for small nanoAB was 0.149 μm.

Big nanoAB was produced as follows. 10 mg ABRAXANE® was reconstituted in8 mg of Bevacizumab in 500 μL 0.9% saline and incubated for 1 hour atroom temperature. After incubation, big nanoAB was brought to 1 mL with0.9% saline. Big nanoAB was further diluted, and 100 μL was administeredto mice for a 24 mg/kg bevacizumab and 30 mg/kg ABRAXANE® dose. Averageparticle size for big nanoAB was 0.226 μm.

Anti-tumor outcomes were statistically superior in mice treated withnanoAB (small and big nanoAB) from the standpoint of tumor volumereduction and survival (FIGS. 23A-C for Group I, 24A-C for Group II, and25A-C for Group III). In addition, histologic (formalin fixed paraffinembedded) analysis of necropsied organs in mice receiving nanoAB therapydid not reveal any unusual toxicities.

In another experiment, female athymic nude mice were injected with 1×10⁶A375 melanoma cells. Tumors were allowed to grow, and treatments wereadministered when tumors were between 600 and 1000 mm³. Mice weretreated intravenously with (a) 100 μL PBS, (b) Bevacizumab (24 mg/kg)only, (c) ABRAXANE® (30 mg/kg) only, (d) Bevacizumb (24 mg/kg) followedthe next day by ABRAXANE® (30 mg/kg) (BEV+ABX), (e) ABRAXANE® (30 mg/kg)followed the next day by Bevacizumb (24 mg/kg) (ABX+BEV), or (f)ABRAXANE®/AVASTIN® complexes with an average diameter of about 0.225 μmin which ABRAXANE® (10 mg/mL) was premixed with Bevacizumb (8 mg/mL) andincubated for 30 minutes before injection (big nanoAB). The percentchange is tumor size at seven days was calculated as follows: [(size onday 7−size on day of treatment)/size on day of treatment]*100. Inaddition, mice were sacrificed when tumors were 2500 mm³ or at 60 daysif tumor size never reached 2500 mm³.

No significant difference was observed between the BEV+ABX and ABX+BEVgroups (FIGS. 28-29). These results suggest that the order of drugadministration does not impact tumor response seven days aftertreatment. The mice treated with ABRAXANE®/AVASTIN® complexes, however,exhibited significant differences as compared to the other treatmentarms with profound tumor response in all mice at day 7 after treatment(FIGS. 28-29). These results demonstrate that ABRAXANE®/AVASTIN®complexes with an average diameter between about 0.15 μm and about 0.3μm can be used successfully to reduce the number of cancer cells withina mammal. In another experiment, female athymic nude mice were injectedwith 1×10⁶ A375 melanoma cells. Tumors were allowed to grow, andtreatments were administered when tumors were between 600 and 1000 mm³.Mice were treated intravenously with (a) 100 μL PBS, (b) Bevacizumab (45mg/kg) only, (c) ABRAXANE® (30 mg/kg) only, (d) Bevacizumb (45 mg/kg)followed the next day by ABRAXANE® (30 mg/kg) (BEV+ABX), (e) ABRAXANE®(30 mg/kg) followed the next day by Bevacizumb (45 mg/kg) (ABX+BEV), or(f) ABRAXANE®/AVASTIN® complexes with an average diameter of about 0.160μm (nanoAB 160), 0.225 μm(nanoAB 225), 0.560 μm (nanoAB 560), or 1.130μm (nanoAB 1130). The nanoAB 160 complexes were prepared by incubating10 mg of ABRAXANE® in 4 mg/mL of AVASTIN®; the nanoAB 225 complexes wereprepared by incubating 10 mg of ABRAXANE® in 8 mg/mL of AVASTIN®; thenanoAB 560 complexes were prepared by incubating 10 mg of ABRAXANE® in10 mg/mL of AVASTIN®; and the nanoAB 1130 complexes were prepared byincubating 10 mg of ABRAXANE® in 15 mg/mL of AVASTIN®. The mixturesincubated for 60 minutes at room temperature and diluted prior toinjection. The percent change is tumor size at seven days was calculatedas follows: [(size on day 7−size on day of treatment)/size on day oftreatment]*100. In addition, mice were sacrificed when tumors were 2500mm³ or at 60 days if tumor size never reached 2500 mm³.

On day 7 post treatment, the mice treated with nanoAB 225, 560, or 1130exhibited tumors with significantly smaller tumor size as compared toall the other treatment groups (FIG. 30). The nanoAB 160 treatment groupwas significantly different than the PBS, BEV only, and ABX only groups,but not statistically different from the sequential administrationgroups, BEV+ABX and ABX+BEV. There were no significant differencesbetween the different nanoAB groups, although the nanoAB 1130 group andthe nanoAB 160 group approached significance (p=0.0952). All mice in thenanoAB 225, 580 and 1130 groups exhibited tumor regression at day 7 posttreatment (FIG. 30). The nanoAB 160 group had 5 of 7 mice exhibit tumorregression at day 7.

These results demonstrate that ABRAXANE®/AVASTIN® complexes with alarger average diameter (e.g., greater than 0.2 μm such as between 0.2μm and 0.9 μm or between 0.2 μm and 1.5 μm) can be more effective thanABRAXANE®/AVASTIN® complexes with a smaller average diameter (e.g., lessthan 0.2 μm such as between 0.05 μm and 0.190 μm or between 0.1 μm and0.190 μm) at seven days post treatment.

Tumor size over time was also assessed (FIG. 31). A delay in tumorgrowth was observed in all mice treated with nanoAB particles, andseveral mice treated with nanoAB particles experienced complete tumorregression (FIG. 31). Survival data also revealed an improvement formice treated with ABRAXANE®/AVASTIN® complexes (FIG. 32). In thisexperiment, while there was no survival advantage for mice treated withnanoAB 160, the remaining nanoAB treatment groups exhibited increasedsurvival as the average particle six of the nanoAB particles increasedwith nanoAB median survival being 30.5, 31.5, and 36 days for nanoAB225, 580, and 1130, respectively. In addition, these survival datademonstrate a survival advantage for mice treated with BEV+ABX (25 days)as opposed to ABX+BEV (20 days). These results also suggest that largerABRAXANE®/AVASTIN® complexes may last longer in circulation or result ina higher deposition of drug in the tumor, thereby resulting in increasedtumor regression.

Example 10 ABRAXANE®/AVASTIN® Complexes as Targeted Therapy for MelanomaPatient Eligibility

The following items are used as inclusion criteria: age ≧18 years,histologic proof of surgically unresectable stage IV malignant melanoma,at least one prior systematic therapy in the metastatic setting that isnot an angiogenesis inhibitor, and measurable disease defined as atleast one lesion whose longest diameter can be accurately measured as≧2.0 cm with chest x-ray or as ≧1.0 cm with CT scan, MRI scan, or CTcomponent of a PET/CT. Disease that is measurable by physicalexamination only is not eligible. Additional inclusion criteria are thefollowing laboratory values obtained 14 days prior to registration:hemoglobin>9.0 gm/dL (patients may be transfused to meet Hgbrequirement), ANC≧1500/mm³, PLT≧100,000/mm³, total bilirubin 1.5 upperlimit of normal (ULN), SGOT (AST)≦2.5×ULN Creatinine 51.5×ULN,Creatinine 1.5×ULN, and an absence of proteinuria at screening asdemonstrated by urine protein/creatinine (UPC) ratio <1.0 at screeningor urine dipstick for proteinuria<2+. Patients discovered to have ≧2-1proteinuria on dipstick urinalysis at baseline should undergo a 24 hoururine collection and demonstrate ≦1 g of protein in 24 hours to beeligible. Additional inclusion criteria are the following: an ECOGPerformance Status (PS) of 0, 1, or 2, the ability to understand and thewillingness to sign a written informed consent document, a willingnessto return to enrolling institution for follow-up (during the activemonitoring phase of the study), a life expectancy≧84 days (3 months), awillingness to provide tissue and blood samples for correlative researchpurposes, and a negative pregnancy test done≦7 days prior toregistration, for women of childbearing potential only.

Exclusion criteria include a known standard therapy for the patient'sdisease that is potentially curative or definitely capable of extendinglife expectancy, prior therapy with an angiogenesis inhibitor, anyanti-cancer therapy or investigational agents ≦4 weeks prior toregistration, uncontrolled intercurrent illness including, but notlimited to, ongoing or active infection, symptomatic congestive heartfailure, unstable angina pectoris, cardiac arrhythmia, or psychiatricillness/social situations that would limit compliance with studyrequirements, a failure to fully recover from acute, reversible effectsof prior chemotherapy regardless of interval since last treatment, orbrain metastases per MRI or CT at any time prior to registration(patients that have had primary therapy for brain metastasis, i.e.,surgical resection, whole brain radiation, or SRT even if stable, arenot eligible). Exclusion criteria also include any of the following:pregnant women, nursing women, and men or women of childbearingpotential who are unwilling to employ adequate contraception. Exclusioncriteria also include co-morbid systemic illnesses or other severeconcurrent disease which, in the judgment of the investigator, wouldmake the patient inappropriate for entry into this study or interferesignificantly with the proper assessment of safety and toxicity of theprescribed regimens, other active malignancy ≦3 years prior toregistration (exceptions: non-melanotic skin cancer or carcinoma-in-situof the cervix. If there is a history or prior malignancy, they must notbe receiving other specific treatment for their cancer), other medicalconditions including, but not limited to, history of liver disease suchas cirrhosis, chronic active hepatitis, chronic persistent hepatitis orhepatitis B or C; active infection requiring parenteral antibiotics;immunocompromised patients and patients known to be HIV positive andcurrently receiving antiretroviral therapy (Patients known to be HIVpositive, but without clinical evidence of an immunocompromised state,are eligible for this trial); New York Heart Association class II-IVcongestive heart failure (Serious cardiac arrhythmia requiringmedication); myocardial infarction or unstable angina ≦6 months prior toregistration; congestive heart failure requiring use of ongoingmaintenance therapy for life-threatening ventricular arrhythmias;clinically significant peripheral vascular disease; history of CNSdisease (e.g., primary brain tumor, vascular abnormalities, etc.),clinically significant stroke or TIA ≦6 months prior to registration,seizures not controlled with standard medical therapy; or history ofhypertensive crisis or hypertensive encephalopathy.

The test schedule is performed as set forth in Table 3.

TABLE 3 Test Schedule Active Monitoring Phase Prior to each ≦21 days ≦14days Cycle 1 sub- Tests and prior to prior to Day Day Day sequentprocedures registration registration 1 8 15 cycle History and X X exam,wt, PS Height X Adverse event X X X X assessment Hematology X X groupWBC ANC Hgb PLT Chemistry group X X X X (AST, total bili, Alk Phos,Creatinine, potassium, sodium, LDH) Serum X pregnancy test¹ Tumor X X ²Measurement/ Evaluation of indicator lesion (CT, MRI, etc.) Mandatoryblood X ³ specimens Mandatory tissue X ⁴ specimens, post dose 1/ cycle 1only ¹For women of childbearing potential only. Must be done ≦7 daysprior to registration. ²Evaluations are performed on day 28 (+/− 3 days)of cycles 2, 4, 6, . . . until disease progression. The same imagingmodality is used throughout the study. ³Blood specimens for PK studies(cycle 1 dose 1, only) are collected in an inpatient facility prior totreatment with ABRAXANE ®/AVASTIN ® complexes, immediately aftertreatment, and every 4 hours for a total of 48 hours. At 24 and 48hours, patients also undergo a CBC and chemistry group blood test toasses for toxicity. Study blood tests for PK analysis are collectedprior to each treatment with ABRAXANE ®/AVASTIN ® complexes during cycle#1 (day 8 and 15). ⁴Study tissue specimens are collected between 20 and26 hours after dose 1/cycle 1 of therapy with ABRAXANE ®/AVASTIN ®complexes while the patients are hospitalized in an in-patient facility.Patients undergo ultrasound or CT guided (radiologist's discretion) 18 gcore needle biopsy (3 passes). One core is collected and processed forparaffin embedding (FFPE); the other 2 cores are snap frozen forpaclitaxel quantification.Protocol Treatment with ABRAXANE®/AVASTIN® Complexes

Actual weight or estimated dry weight if fluid retention is used. Thetreatment schedule for ABRAXANE®/AVASTIN complexes is repeated eachmonth (every 28 days +/−3 days) or until disease progression, patientrefusal, or unacceptable toxicity (Table 4) with the indicated doseescalation scheme (Table 5) and dose limiting toxicities (Table 6).

TABLE 4 Agent Dose Route Days ReRx ABRAXANE ®/ assigned at time IV over60 minutes (only 1, 8 and 15 Every 28 AVASTIN ® of registration 1^(st)dose; subsequent doses days* complexes infused over 30 minutes) *Onetreatment cycle = 28 days +/− 3 days

TABLE 5 Dose Escalation Scheme. Dose Level Dose (ABX) Dose (BEV) −2  75mg/m² 30 mg/m² −1 100 mg/m² 40 mg/m²    1* 125 mg/m² 50 mg/m²   2 150mg/m² 60 mg/m²   3 175 mg/m² 70 mg/m² *Starting dose.

TABLE 6 Dose Limiting Toxicities (DLT). Toxicity DLT DefinitionHematologic Grade 4 ANC, Grade 4 Hgb, or PLT <25,000 Renal Serumcreatinine ≧2 times baseline Other nonhematologic ≧ grade 3 as per NCICommon Terminology Criteria for Adverse Events (CTCAE) version 4.0

Determination of Maximum Tolerated Dose (MTD)

The maximum tolerated dose is defined as the highest dose level amongthose tested where at most one out of six patients develops a DLT priorto the start of their second cycle of treatment and the next highestdose level is such that two out of a maximum of six patients treated atthis dose level developed a DLT prior to the start of their second cycleof treatment.

Enrollment and Determination of MTD

A minimum of two or a maximum of six patients are accrued to a givendose level. For dose level 1 (and if accrued to, dose levels −1 & −2),enrollment is temporarily halted after each patient has been enrolled inorder to gather acute adverse event data over the first cycle of theirtreatment. For dose levels 2 & 3, patients are accrued to these doselevels so that at any given time no more than two patients are receivingtheir first cycle of treatment and acute adverse event data over thefirst treatment cycle for all other patients treated at the current doselevel is known. If, at any time in the enrollment process, two patientstreated at the current dose level develop a DLT during the first cycleof treatment, enrollment is closed to that dose level. Enrollment isre-opened to the next lower dose level if fewer than six patients havebeen treated at that dose level. If none of the first three patientstreated at a given dose level develops a DLT during the first cycle oftreatment, enrollment to the dose level is closed and enrollment isreopen at next higher dose level. If there are no other higher doselevels to be tested, three additional patients are enrolled at thecurrent dose level to confirm MTD. If one of the first three patientstreated at a given dose level develops a DLT during the first cycle oftreatment, three additional patients are enrolled (sequentially) ontothe current dose level. If, at any time in the enrollment of these threeadditional patients, a patient develops a DLT, enrollment is closed tothis dose level. Enrollment is re-opened to the next lower dose level iffewer than six patients are treated at that dose level. If none of thesethree additional patients develops a DLT during the first cycle oftreatment, enrollment to this dose level is closed and enrollment isreopened at next higher dose level. If there are no other higher doselevels to be tested, this is considered the MTD.

For this protocol, the patient returns for evaluation and retreatment(at least every 28 +/−3 days) according to the schedule. If a patientfails to complete the first cycle of treatment for reasons other thantoxicity, an additional patient is enrolled to replace this patient.

Dosage Modification Based on Adverse Events

The modifications in Table 7 are strictly followed until individualtreatment tolerance is ascertained. If multiple adverse events (Table 8)are seen, dose is administered based on greatest reduction required forany single adverse event observed. Dose modifications apply to thetreatment given in the preceding cycle and are based on adverse eventsobserved since the prior dose.

TABLE 8 Use Common Terminology Criteria for Adverse Events (CTCAE) v.4.0* unless otherwise specified CTCAE Category Adverse Event DoseReduction Investigations ANC <1000 Day 1: Hold until counts above theselevels. or Day 8: Omit dose that day and retreat at same dose PLT<75,000 level on day 15 if counts have recovered. Day 15: Omit dose thatday. NOTE: if two consecutive cycles of therapy require omission of adose, subsequent treatment cycles should begin (day 1) at next lowerdose. AST or Day 1: Hold until resolved to <Grade 2 then reduce Alkalinedose by ONE dose level. Phosphatase If treatment needs to be held >4weeks, discontinue ≧ Grade 2 study treatment and go to event monitoring.Neurology Neuropathy Day 1: Hold until resolved to <Grade 2 then reducedisorders ≧ Grade 2 dose by ONE dose level. Day 8 OR 15 - Omit dose thatday. If resolved to <Grade 2 by next scheduled dose, then dose reduce byone level If treatment needs to be held >4 weeks, discontinue studytreatment and go to Event Monitoring All other non- ≧ Grade 3 Day 1:Hold until resolved to ≦ Grade 2 then reduce hematologic dose by ONEdose level. adverse events Day 8: Omit dose that day. If resolved to ≦Grade 2 by day 15, then dose reduce by one level and retreat. Day 15:Omit dose that day. NOTE: if two consecutive cycles of therapy requireomission of a dose, subsequent treatment cycles should begin (day 1) atnext lower dose. If treatment needs to be held >4 weeks, discontinuestudy treatment and go to Event Monitoring Gastrointestinal BowelDiscontinue all study treatment and proceed to Disorders perforationEvent Monitoring Bowel Continue patient on study for partial bowelObstruction obstruction NOT requiring medical intervention. Grade 1 Holdfor partial obstruction requiring medical Grade 2 intervention. Ifresolved to Grade 0 within 4 weeks, Grade 3 or 4 treatment may berestarted. If treatment needs to be held >4 weeks, discontinue all studytreatment and go to Event Monitoring. For complete bowel obstruction,discontinue study treatment and proceed to Event Monitoring CardiacDisorders Hypertension Hypertension should be treated as per general≧Grade 3 practice. If hypertension (≧150/100) persists despitetreatment, hold treatment until blood pressure is below this level Iftreatment needs to be held >4 weeks due to uncontrolled hypertension,discontinue study treatment and go to Event Monitoring. Left ventricularHold until resolution to Grade ≦1. If treatment systolic needs to beheld >4 weeks, discontinue all study function- treatment and go to EventMonitoring. Grade 3 Discontinue treatment and proceed to Event Grade 4Monitoring Respiratory, Bronchopulmonary Discontinue all study treatmentand proceed to thoracic and Hemorrhage Event Monitoring mediastinal ≧Grade 2 disorders Coagulation Hemorrhage Hold until ALL of the followingcriteria are met: Grade 3 1. Bleeding has resolved and Hb is stable.Grade 4 2. There is no bleeding diathesis that would increase the riskof therapy. 3. There is no anatomic or pathologic condition that couldincrease the risk of hemorrhage recurrence. If treatment needs to beheld >4 weeks, discontinue study treatment and go to Event MonitoringPatients who experience a recurrence of Grade 3 hemorrhage are todiscontinue all study treatment and proceed to Event Monitoring.Discontinue study treatment and proceed to Event Monitoring BleedingDiscontinue study treatment and proceed to Event diathesis MonitoringGrade 3 or 4 Vascular disorders Venous Hold treatment. If the plannedduration of full- thrombosis dose anticoagulation is <2 weeks, treatmentshould Grade 3 be held until the full-dose anticoagulation period or isover. asymptomatic If the planned duration of full-dose Grade 4anticoagulation is >2 weeks, treatment may be Symptomatic resumed duringthe period of full-dose Grade 4 anticoagulation IF all of the criteriabelow are met: The subject must have an in-range INR (usually 2-3) on astable dose of warfarin, or on stable dose of heparin prior torestarting treatment. The subject must not have pathological conditionsthat carry high risk of bleeding (e.g. tumor involving major vessels orother conditions) The subject must not have had hemorrhagic events whileon study If thromboemboli worsen/recur upon resumption of study therapy,discontinue treatment. Discontinue treatment and proceed to EventMonitoring Arterial Discontinue treatment and proceed to Eventthrombosis Monitoring (Angina, myocardial infarction, transient ischemicattack, cerebrovascular accident, or any other arterial thromboembolicevents) ANY Grade

Ancillary Treatment/Supportive Care

Routine use of colony-stimulating factors (G-CSF or GM-CSF) is notrecommended. Prophylactic use of colony-stimulating factors during thestudy is not allowed. Therapeutic use in patients with seriousneutropenic complications such as tissue infection, sepsis syndrome,fungal infection, etc., may be considered at physician discretion.Recombinant erythropoietin to maintain adequate hemoglobin levels andavoid packed red blood cell transfusions is allowed.

Patients should receive full supportive care while on this study. Thisincludes blood product support, antibiotic treatment and treatment ofother newly diagnosed or concurrent medical conditions. All bloodproducts and concomitant medications such as antidiarrheals, analgesics,and anti-emetics received from the first administration of study drugsuntil 30 days after the final dose arc to be recorded in the medicalrecord. Patients participating in phase I program clinical trials arenot to be considered for enrollment in any other study involving apharmacologic agent-(drugs, biologics, immunotherapy approaches, genetherapy) whether for symptom control or therapeutic intent.

Hypersensitivity Reactions

Patients do not require premedication prior to administration ofABRAXANE®/AVASTIN® complexes. In the unlikely event of ahypersensitivity reaction, treatment with antihistamines, H2 blockers,and corticosteroids is recommended. Patients should be pre-medicatedwith the typical regimen for paclitaxel regimens for subsequent cycles.In the unlikely event of a mild hypersensitivity reaction, premedicationmay be administered using the premedication regimen the institutiontypically uses for solvent-based paclitaxel.

ABRAXANE®/AVASTIN® Complexes

ABRAXANE®/AVASTIN® complexes are prepared as a hazardous low riskproduct. ABRAXANE® is supplied as a white to off-white lyophilizedpowder containing 100 mg of paclitaxel and approximately 900 mg AlbuminHuman USP (HA) as a stabilizer in a 50 mL, single-use vial. Each vial ofthe lyophilized product is reconstituted as set forth below.Unreconstituted ABRAXANE® is stored at controlled room temperature inits carton. Reconstituted ABRAXANE® is used immediately. AVASTIN®(bevacizumab) is classified as an anti-VEGF monoclonal antibody and avascular endothelial growth factor (VEGF) inhibitor. AVASTIN® issupplied in 100 mg (4 mL) glass vials, with a concentration of 25 mg/mL.Vials contain AVASTIN® typically with phosphate, trehalose, polysorbate20, and sterile water for injection (SWFI), USP. Vials contain nopreservative and are suitable for single use only. AVASTIN® vials arestored in a refrigerator at 2° C.-8° C. AVASTIN® vials are kept in theouter carton due to light sensitivity. They are not frozen or shaken.

Chemical and physical in-use stability of AVASTIN® is acceptable for 48hours at 2° C.-30° C. in 0.9% sodium chloride solution. AVASTIN® is notadministered or mixed with dextrose solution. AVASTIN® is furtherdiluted as set forth below.

The ABRAXANE®/AVASTIN® complexes arc prepared as a hazardous low riskproduct. The dose appropriate number of 4 mL vials of 25 mg/mL AVASTIN®(bevacizumab) are obtained, and each vial is further diluted per thefollowing directions to 4 mg/mL. The dose appropriate number ofABRAXANE® (paclitaxel) 100 mg vials is obtained and each vial isreconstituted per the following directions to a final concentrationcontaining 10 mg/mL nanoparticic albumin-bound (nab) paclitaxel. It isnot a requirement to use filter needles in the preparation of, orin-line filters during administration. In addition, filters of pore-sizeless than 15 micrometers are to be avoided.

As with other cytotoxic anticancer drugs, caution is exercised inhandling ABRAXANE®. The use of gloves is recommended.

Using a sterile 3 mL syringe, 1.6 mL (40 mg) of AVASTIN® 25 mg/mL iswithdraw and slowly injected, over a minimum of 1 minute, onto theinside wall of each of the vials containing 100 mg of ABRAXANE®. UnusedAVASTIN ® left in the 25 mg/mL vial is discarded, as the productcontains no preservatives. Injecting the AVASTIN® solution directly ontothe lyophilized cake is avoided as this will result in foaming. Using asterile 12 mL sterile syringe, 8.4 mL of 0.9% Sodium Chloride

Injection, USP, is withdraw and slowly injected, over a minimum of 1minute, onto the inside wall of each vial containing ABRAXANE® 100 mgand AVASTIN® 40 mg. Once the addition of AVASTIN® 1.6 mL and 0.9% SodiumChloride Injection, USP 8.4 mL is complete in each vial, each vial isgently swirled and/or inverted slowly for at least 2 minutes untilcomplete dissolution of any cake/powder occurs. The generation of foamis avoided. The concentration of each vial is 100 mg/10 mL ABRAXANE and40 mg/10 mL AVASTIN®. The vials containing the ABRAXANE® and AVASTIN®are allowed to sit for 60 minutes. The vial(s) are gently swirled and/orinverted every 10 minutes to continue to mix the complexes. After 60minutes is elapsed, a sterile 60- to 100-mL syringe (appropriate sizefor the volume being administered) is used to withdraw the calculateddosing volume of ABRAXANE® and AVASTIN® from each vial. A sufficientquantity of 0.9% Sodium Chloride Injection, USP is added to make thefinal concentration of ABRAXANE® 5 mg/mL and AVASTIN® 2 mg/mL. Thesyringe is gently swirled and/or inverted slowly for 1 minute to mix.The storage and stability is for up to 4 hours at room temperaturefollowing final dilution.

Administration

The IV initial complex dose is infused over 60 minutes via syringe pump.The infusion may be shortened to 30 minutes if the initial infusion iswell tolerated. Infusion is monitored closely during the infusionprocess for signs/symptoms of an infusion reaction. The patient's lineis flushed after administration with 20 mL 0.9% Sodium Chloride. Anexample calculation and preparation is as follows:

-   -   Dose level 1: ABRAXANE® 125 mg/m² /AVASTIN® 50 mg/m² BSA=2 m²    -   Doses required: ABRAXANE® 250 mg/AVASTIN® 100 mg    -   Obtain three 100 mg vials of ABRAXANE®.    -   Obtain one 100 mg vial of AVASTIN® 25 mg/mL.    -   Withdraw 1.6 mL (40 mg) of AVASTIN® 25 mg/mL and slowly inject        over 1 minute onto the inside wall of one of the 100 mg        ABRAXANE® vials. Repeat this procedure for each of the remaining        two ABRAXANE® 100 mg vials.

Add 8.4 mL 0.9% Sodium Chloride Injection, USP onto the inside wall ofone of the vials containing ABRAXANE® and AVASTIN®. Repeat thisprocedure for each of the remaining two ABRAXANE® and AVASTIN® vials.

Let mixture sit for 60 minutes (swirling every 10 minutes). The finalconcentration of each vial should be 100 mg ABRAXANE®/10 mL and 40 mgAVASTIN®/10 mL.

Withdraw 25 mL from the ABRAXANE® and AVASTIN® containing vial and placein a 100 mL sterile syringe. Add 25 mL 0.9% Sodium Chloride Injection,USP for a final ABRAXANE® concentration of 5 mg/mL and AVASTIN®concentration of 2 mg/mL. Infuse via syringe pump over 60 minutes (firstdose, 30 minutes subsequent doses).

Response to ABRAXANE®/AVASTIN® Complex Treatment

Each patient's response to treatment with a ABRAXANE®/AVASTIN® complexformulation is monitored.

Example 11 Making ABRAXANE®/AVASTIN® Complexes

ABRAXANE® was incubated with various increasing concentrations ofAVASTIN® to form ABRAXANE®/AVASTIN® complexes of increasing diameter.Ten milligrams of ABRAXANE® was reconstituted in 1 mL of AVASTIN® at 0,2, 4, 6, 8, 10, 15, and 25 mg/mL, and the mixture was incubated at roomtemperature for 30 minutes. After incubation, the distributions ofparticle sizes were determined with the Mastersizer 2000. The medianparticle size ranged from 0.146 μm to 2.166 μm for 0 and 25 mg/mLAVASTIN®, respectively (FIG. 25). It appeared that as AVASTIN®concentrations increased, the particles formed singlets, doublets, andtetramers. These results demonstrate that the antibody concentration inwhich ABRAXANE® is incubated impacts the size of the nanoparticle. Asdemonstrated herein, manipulating the size of the particles can changethe pharmacokinetics of the drug complex as well as itsbio-distribution, which in turn can improve the clinical efficacy of thedrug complex.

Example 12 Making ABRAXANE®/Rituxan® Complexes

ABRAXANE® was incubated with various increasing concentrations ofRituxan® (rituximab) to form ABRAXANE®/Rituxan® complexes of increasingdiameter. Ten milligrams of ABRAXANE® was reconstituted in 1 mL ofRituxan® at 0, 2, 4, 6, 8, and 10 mg/mL, and the mixture was incubatedat room temperature for 30 minutes. After incubation, the distributionsof particle sizes were determined with the Mastersizer 2000. The medianparticle size ranged from 0.147 μm to 8.286 μm for 0 and 10 mg/mLRituxan®, respectively (FIG. 26). These results demonstrate that theantibody concentration in which ABRAXANE® is incubated impacts the sizeof the nanoparticle. These results also demonstrate that differenthumanized therapeutic antibodies can result in different particle sizeswhen mixed with ABRAXANE® at the same concentration. As demonstratedherein, manipulating the size of the particles can change thepharmacokinetics of the drug complex as well as its bio-distribution,which in turn can improve the clinical efficacy of the drug complex.

Example 13 Making ABRAXANE®/Herceptin® Complexes

ABRAXANE® was incubated with various increasing concentrations ofHerceptin® (also referred to as rituximab or trastuzumab) to formABRAXANE®/Herceptin® complexes of increasing diameter. Ten milligrams ofABRAXANE® was reconstituted in 1 mL of IIerceptin® at 0, 2, 4, 6, 8, 10,15, and 22 mg/mL, and the mixture was incubated at room temperature for30 minutes. After incubation, the distributions of particle sizes weredetermined with the Mastersizer 2000. The median particle size rangedfrom 0.147 μm to 2.858 μm for 0 and 22 mg/mL Herceptin®, respectively(FIG. 27). These results demonstrate that the antibody concentration inwhich ABRAXANE® is incubated impacts the size of the nanoparticle. Theseresults also demonstrate that different humanized therapeutic antibodiescan result in different particle sizes when mixed with ABRAXANE® at thesame concentration. As demonstrated herein, manipulating the size of theparticles can change the pharmacokinetics of the drug complex as well asits bio-distribution, which in turn can improve the clinical efficacy ofthe drug complex.

Example 14 Dissociation Constants

The association and dissociation of AVASTIN with human serum albumin andABRAXANE® were determined. In this experiment, biotinylated AVASTIN® wasbound to a streptavidin sensor. After AVASTIN® loading to the sensor,the sensor was exposed to either 1 mg/mL of human serum albumin or 1mg/mL of ABRAXANE®. This experiment demonstrated that AVASTIN® binds toboth human serum albumin and ABRAXANE®. The dissociation constants werecalculated to be 6.2×10⁻⁶ and 5.873×10⁻⁷ for human serum albumin andABRAXANE®, respectively.

The association and dissociation of albumin with AVASTIN® and AVASTIN®with ABRAXANE® were determined. In this experiment, biotinylatedAVASTIN® or biotinylated albumin® was bound to a streptavidin sensor.After albumin or AVASTIN® loading to the sensor, the sensor was exposedto 1 mg/mL of AVASTIN® or 1 mg/mL of ABRAXANE®, respectively. Thisexperiment demonstrated that albumin binds to AVASTIN® and AVASTIN®binds to ABRAXANE®. The dissociation constant calculated for albumin andAVASTIN® was 6.588×10⁻¹⁰. The dissociation constant calculated forAVASTIN® and ABRAXANE® in this experiment was 1.698×10⁻⁵.

Example 15 ABRAXANE®/AVASTIN®/Cisplatin Complexes Inhibit Tumor CellProliferation

Proliferation of A375 melanoma tumor cells in vitro following exposureto various treatments was assessed. Briefly, cells were exposed toincreasing concentrations of (a) ABRAXANE® only (ABX; 0-1000 μg/mL), (b)cisplatin only (0-200 μg/mL), (c) ABRAXANE®/AVASTIN® complexes with anaverage diameter of 0.155 (nanoAB; 0-1000 μg/mL), orABRAXANE®/AVASTIN®/cisplatin complexes with an average diameter of0.141. Cisplatin is a chemotherapy drug that is highly effective againsttumors, but has such a high toxicity to normal tissue that it isinfrequently used in the clinic. One can appreciate the high toxicity ofcisplatin alone in that 100% of cells are killed at 100 μg/mL (FIG. 33).With reference to FIG. 33, the complexes were made with an ABRAXANE®:AVASTIN® at a 2.5 to 1 ratio. The x-axis numbers refer to only thepaclitaxel and cisplatin concentrations with the higher number being thepaclitaxel concentration and the other being cisplatin. The range ofdoses were different because cisplatin is so toxic. To make theABRAXANE®/AVASTIN®/cisplatin complexes, ABRAXANE® (10 mg/mL), AVASTIN®(4 mg/mL), and cisplatin (2 mg/mL) were co-incubated at room temperaturefor 30 minutes. The nanoparticles were spun for 10 minutes at 5000 rpmto remove unbound cisplatin. The nanoparticles were resuspended in 0.9%saline and added to the wells containing 50,000 A375 cells. The cellswere incubated overnight at 37° C. The cells were stained with athymidine analog, EdU, which incorporates into the DNA as cellsproliferate. Cells that are actively proliferating stain positive. Theproliferation index was calculated as the % positive cells in treatedcells/% positive cells in untreated cells. This resulted in an index ofthe number of proliferating cells for treatment relative to the highestreading of % positive for untreated cells.

Cisplatin was determined to be present in the nanoABC particles due tothe increase in drug toxicity relative to ABRAXANE® alone and nanoAB(FIG. 33). The toxicity of nanoABC, however, was not as high ascisplatin only, suggesting that cisplatin may be used in the complex toincrease drug toxicity to the tumor while having limited toxicity tonormal tissue.

The particle size distributions for the ABRAXANE® particles, theABRAXANE®/AVASTIN® complexes (nanoAB), and theABRAXANE®/AVASTIN®/cisplatin complexes (nanoABC) used above weredetermined. The median size of the particles was 0.146 μm, 0.155 μm, and0.141 μm, for ABX, nanoAB, and nanoABC, respectively (FIG. 34). Theseresults demonstrate that particle size when cisplatin is added was notdifferent from the particle size when only ABRAXANE® and AVASTIN® arepresent.

Example 16

Treating Cancer with ABRAXANE®/AVASTIN®/Cisplatin Complexes

Athymic nude mice were injected with 1×10⁶ A375 human melanoma tumorcells. The tumors were allowed to grow, and when the tumors were 600 to1000 mm³, the mice were treated intravenously with PBS, ABRAXANE® (30mg/kg), cisplatin (2 mg/kg), nanoAB160 (30 mg/mL ABX and 8 mg/mL BEV),nanoAB160 and cisplatin at the same concentrations as above, and nanoABC(30 mg/kg ABX, 8 mg/kg BEV, and 2 mg/kg Cis). NanoABC was prepared asfollows: 10 mg of ABRAXANE® was reconstituted in 4 mg/mL bevacizumab and2 mg/mL cisplatin and allowed to incubate at room temperature for 30minutes. Following incubation, the complexes were diluted for mouseinjection. Mice were treated once, and tumor growth was monitored for atleast 80 days for all mice.

Tumor growth kinetics among the treatment groups demonstrated delayedtumor growth in three groups: nanoAB160 (AB Complex), nanoAB160+Cisplatin, and nanoABC (ABC Complex) (FIGS. 35-37). There was onecomplete response in each of the nanoAB (1/7, 14%) and nanoAB cisplatin(1/7, 14%) groups, and two complete responses in the nanoABC group (2/7,28.5%). At day 7 post-treatment, 20 of 21 (95%) mice in the three groupsreceiving a nanoparticle demonstrated a tumor response while 0 of 15mice had tumor responses in the control groups. The nanoABC group hadthe highest median survival at 35 days. The other groups median survivalwas 8, 15, 9, 24, and 26 days for PBS, ABRAXANE , cisplatin, nanoAB160,and nanoAB160 plus cisplatin, respectively.

Example 17 Heat Stability

To measure nanoAB stability, ABRAXANE® and bevacizumab were directlylabeled with the fluorescent markers FITC and APC, respectively, as perthe manufacturer's instructions (Thermo Scientific). Unincorporatedlabel was removed by size filtration on a sepharose column. Once labeledABRAXANE® and bevacizumab were incubated together to form complexes asdescribed herein. The complexes were then run on a flow cytometer(Guava, Millipore), and data was analyzed using Guava Incyte software.The stability of the complexes at room temperature in PBS was assessed(FIG. 38). The percentage of complexes double positive for ABRAXANE® andbevacizumab was 75% at time 0, and the percentage at 24 hours was 72%,demonstrating that the complexes were highly stable at room temperature.

The complexes also were incubated at 37° C. in human plasma (FIG. 39).The data indicated that the complexes break down within 30 minutes inplasma at 37° C. and only about 13% remains in the large complex. Thepercentage shown in FIG. 39 are the percentages of complexes stayingconstant after 30 minutes for at least 2 hours. In a similar experiment,the complexes were incubated at 37° C. for up to 3 hours (FIG. 40).After incubation, the samples were run on a polyacrylamide gel toseparate proteins by size and Western blotted with an anti-taxolantibody. These results suggest that while the big complexes break downin human plasma at 37° C., the break down product was about a 200 kDprotein that contains albumin, bevacizumab, and paclitaxel.

Example 18 Binding Characteristics

The following was performed to assess protein binding due todeglycosylation of bevacizumab compared to naturally occurring IgG_(—)In order to determine if bevacizumab binding to ABRAXANE® is due to thedeglycosylation of the Fe chain of the antibody, the binding kinetics ofbevacizumab were compare to naturally produced IgG isolated from humanplasma. These experiments suggest that naturally occurring, fullyglycosylated IgG exhibited a higher dissociation constant thanbevacizumab.

Example 19 Nano-Particles and the Use of Nanoparticles to Treat Cancer

The following provides a summary of selected results from the aboveExamples, which in some cases may include results from additionalstudies.

Materials and Methods

AB160 preparation and size estimation: Ten milligrams of nab-paclitaxelpower was reconstituted in 0.9% saline or bevacizumab at the followingconcentrations; 2, 4, 6, 8, 10, 15, and 25 mg/ml. The 1 ml mixtures wereallowed to incubate for 1 hour at room temperature. The size of theparticles was measured by light refraction using a Mastersizer 2000(Malvern Instruments, Worcestershire, UK).

Immunofluorescence imaging of AB160: One hundred microliters ofnab-paclitaxel was mixed with 100 ul of 0.5, 5, 10 and 25 mg/ml ofbeacizumab. The mixtures were incubated for 1 hour at room temperatureand light microscopy pictures were taken at a magnification of 400×. Forconfocal and flow cytometry, nab-paclitaxel was directly labeled withFITC and bevacizumab was labeled with APC according to manufacturer'sinstructions (Thermo Scientific, Rockford, Ill.). Once labeled thenab-paclitaxel and bevacizumab were co-incubated for 30 minutes at roomtemperature and looked at by confocal microscopy (3LSM Confocal, CarlZeiss MicroImaging) and flow cytometry (Guava Easycyte 8HT EMDMillipore). Flow cytometry data was analyzed using GuavaSoft software(EMD Millipore, Billerica, Mass.).

Western blotting: Nab-paclitaxel (45 mg/mL) was mixed 1:1 withbevizcumab at a concentration of 10 mg/mL or 1 mg/mL and incubated 4hours or overnight at room temperature (25° C.). After incubation, themixture was spun at 13,000 RPM for 10 minutes at 4° C. The supernatantwas collected, mixed 1:1 with Laemmli buffer and boiled for 3 minutesprior to being loaded on at 7.5% Tris-HCl criterion gel. The gel was runat 100 volts for 2 hours before it was transferred overnight at 20volts. 5% milk in TBST was used to block the membrane after transfer anda primary anti-mouse (Fab) IgG-HRP (1:1000) and rabbit anti-taxol(1:500) antibody was used to probe the membrane. Membranes were washed 3times for 15 minutes. A secondary anti-rabbit IgG-HRP antibody(1:10,000) was used to label the taxol membrane. The membranes wereagain washed and ECL detection reagent was added to each membrane for 5minutes. Membranes were developed on a Kodak M35A-M X-OMAT Processor andexposed for 1 second (Taxol) or 1 minute (Bevizcumab).

Bevicuzumab was diluted to a concentration of 0.25 mg/mL and hSA wasdiluted to a concentration of 0.05 mg/mL. The two were added 1:1 andincubated for 30 minutes at room temperature. Laemmli buffer was added1:1 with the samples and boiled for 3 minutes without 2 ME. The sampleswere loaded on at 7.5% Tris-HCl criterion gel and run at 100 volts for 2hours before being transferred overnight at 20 volts. 5% milk in TBSTwas used to block the membrane after transfer and a primary anti-humanalbumin (1:10,000) antibody was used to probe the membrane. Membraneswere washed 3 times for 15 minutes. A secondary anti-rabbit IgG-HRPantibody (1:10,000) was used to label the hSA membrane. The membraneswere again washed and ECL detection reagent was added to the membranefor 5 minutes. The membrane was developed on a Kodak M35A-M X-OMATProcessor and exposed for 1 minute.

In vitro AB160 function—proliferation assay: The melanoma cell line,A375, was exposed to nab-paclitaxel alone or AB160 at concentrationsfrom 0 to 200 ug/ml paclitaxel overnight in the presence of EdU, athymidine analog. After the overnight incubation, the A375 cells wereharvested, permeabolized and intracellularly stained with a FITCconjugated anti-EdU antibody. Cell proliferation was determined by DNAsynthesis as a percentage of cells, which were FITC positive on a Guava8HT flow cytometer (Millipore Billerica, Mass.). Data analysis wasperformed using Gauva Incyte software (Millipore Billerica, Mass.). Theproliferation index was calculated by dividing the percentage ofproliferating cells in treated wells (FITC positive) by the percentageof cells proliferating in the untreated well.

In vitro AB160 function—ligand Binding of AB160: High protein binding 96well plates were coated overnight at 4° C. with 5 mg/ml nab-paclitaxel,1.25 mg/ml bevacizumab or AB160 containing 5 mg/ml ABRAXANE plus 1.25mg/ml bevacizumab. The plates were washed 3 times with PBS+0.5%Tween-20. VEGF was added to the drug coated wells at concentrations from0 to 4000 μg/ml and incubated at room temperature for 2 hours. After 2hours the unbound VEGF was removed and measured via standard VEGF ELISA(R and D Systems Minneapolis, Minn.). The percent of drug bound VEGF wascalculated by (concentration VEGF after drug exposure/totalconcentration of VEGF measured from standard curve)*100.

In vitro AB160 function—small animal model: Female athymic nude micewere injected with 1×10⁶ A375 melanoma cells in the flank. Tumors wereallowed to grow, and treatments were administered when tumors werebetween 600 and 1000 mm³. Mice were treated with (a) 100 μL PBS, (b)bevacizumab (8 mg/kg), (c) nab-paclitaxel (30 mg/kg), (d) bevacizumb(day 0 8 mg/kg) followed by nab-paclitaxel (day 1, 30 mg/kg), (e) AB160which was produced as follows: 10 mg nab-paclitaxel was reconstituted in3.6 mg of bevacizumab in 500 μL 0.9% saline and incubated for 1 hour atroom temperature. After incubation, AB160 was brought to 1 mL with 0.9%saline. AB160 was further diluted, and 100 μL was administered to micefor a final 8 mg/kg bevacizumab and 30 mg/kg nab-paclitaxel dose. Tumorsize was monitored 2-3 times per week. Mice were sacrificed when tumorsreached 2000-2500 mm³. Percent change from baseline was calculated by[(tumor size on day 7−tumor size on day of treatment)/tumor size on dayof treatment]*100.

Results

Under specific in vitro mixing conditions, the mixing of varyingconcentrations of clinical grade bevacizumab and nab-paclitaxel createsa range of different size macromolecular complexes as determined bydirect visualization with phase contrast light microscopy (FIG. 9) andlight scatter size distribution analysis (FIG. 25).

Immunofluorescent labeling of bevacizumab and/or nab-paclitaxeldemonstrated double-labeling of the macromolecular complexes usingimmunofluorescence microscopy (FIG. 41A) and flow cytometry (FIG. 41B).These data suggest that in vitro mixing of bevacizumab andnab-paclitaxel in varying relative concentrations results in thecreation of macromolecular complexes of different sizes containing bothdrugs.

With the aim of developing an agent (macromolecular complex) mostamenable to rapid clinical translation (minimal alteration of existingFDA approved agents), efforts were concentrated on furthercharacterizing the bevacizumab/nab-paclitaxel complex demonstrating amedian particle size of 160 nm (AB160). Under the conditions used,approximately 80% of the complex formed the 160 nm particle, and roughly20% consisted of 200 kD molecules containing paclitaxel and bevacizumab(FIG. 42A). The 160 nm diameter of the AB160 complex approximated amonolayer coating of nab-paclitaxel by bevacizumab (FIG. 42B; cartoonand EM image of AB160 stained with anti-human-Ig-gold conjugate).Bevacizumab appeared to bind to nab-paclitaxel at the level of thealbumin mantle of the nanoparticle via its Fe domain retaining VEGFbinding capacity (FIG. 42C). Flow-cytometric analysis of the AB160particle (versus nab-paclitaxel) demonstrated that maximal fluorescenceof the nanoparticles was achieved when a fluorescently labeled anti-VEGFantibody was incubated with the AB160 complex in the presence of VEGF;significantly less anti-VEGF staining was observed for nab-paclitaxelincubated with VEGF alone. This suggested a somewhat unexpected affinityof bevacizumab to albumin that can easily be demonstrated byco-incubating human serum albumin (hSA) and bevacizumab and blotting foralbumin (FIG. 42D). Under non-denaturing conditions, the albumin band,in the bevacizumab/albumin mixture, migrated at a MW of approximately2001 (d, as would be predicted for an albumin/bevacizumab complex (60kD+140 kD, respectively). Affinity analyses of thebevacizumab/nab-paclitaxel and the bevacizumab/hSA complex dissociationconstants (Kd=5.8×10⁻⁷, and Kd=6.2×10⁶, respectively) suggested that thebevacizumab/albumin interaction is hydrophobic. Additionally, the AB160complex retained both the antiproliferative properties of nab-paclitaxelas well as the VEGF binding properties of bevacizumab (FIGS. 43A and43B).

Nude mice implanted with the human A375 melanoma developed tumors withsizes in the range of 1000 mm³ at the time of single IV injection withAB160, bevacizumab, nab-paclitaxel, or sequential infusion ofbevacizumab followed by next-day nab-paclitaxel (FIG. 44). Tumor growthkinetics as well as percent change of tumor size following the singleinjection of drug demonstrated that the most favorable outcomes wereobserved in the AB160 cohort. Pharmacokinetic analysis of peripheralblood and tumors following a single injection of either AB160,nab-paclitaxel or bevacizumab, demonstrated: (a) prolongation of theplasma elimination of bevacizumab in AB160 versus that of bevacizumabalone (FIG. 45A); and (b) significantly increased percentage of tumorcells demonstrating intracellular paclitaxel (by IHC) in the AB160cohort relative to that of nab-paclitaxel alone or saline treatedcontrols (FIG. 45B). Of note, there appeared to be a correlation betweentissue sections that stained positive with paclitaxel and those stainingpositive for human immune globulin (detecting bevacizumab, FIG. 45C, Rvalues of 0.7445 and 0.5496). No such correlations were detected innab-paclitaxel (ABX) alone treated mice (R values of 0.0176 and 0.0229).

Collectively, these data suggest that the AB160 formulation ofnab-paclitaxel allows for prolonged circulation and increased deliveryof paclitaxel at the VEGF expressing tumor site, likely responsible forthe observed “clinical” benefit. In effect, the AB160 macromoleculeseems to increase the efficiency of paclitaxel delivery into the VEGFexpressing malignancy. Ongoing data further support this observation bydescribing the in vivo AB160 dissociation subunits as hetero-trimersconsisting of bevacizumab-albumin-paclitaxel. This is further supportedby the observed improved clinical benefit of larger AB complexes insimilar in vivo A375 mouse xenograft experiments.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications arc within the scope of thefollowing claims.

1. (canceled)
 2. An antibody-albumin nanoparticle complex comprising albumin, an anti-CD52 antibody, and a therapeutically effective amount of paclitaxel, wherein the nanoparticle complex has been pre-formed in vitro by mixing an aqueous albumin-paclitaxel nanoparticle with the antibody under conditions to form the nanoparticle complex, such that the antibody retains anti-CD52 binding specificity, said complex having a diameter less than 1 μm.
 3. The antibody-albumin nanoparticle complex of claim 2, wherein the antibody is a humanized antibody.
 4. The antibody-albumin nanoparticle complex of claim 2, wherein the antibody binds an antigen on a tumor cell.
 5. The antibody-albumin nanoparticle complex of claim 4, wherein the tumor cell is a solid tumor cell.
 6. The antibody-albumin nanoparticle complex of claim 4, wherein the tumor cell is derived from a lymphoma or leukemia.
 7. The antibody-albumin nanoparticle complex of claim 2, further comprising an alkylating agent.
 8. The antibody-albumin nanoparticle complex of claim 7, wherein said alkylating agent is a platinum compound.
 9. The antibody-albumin nanoparticle complex of claim 8, wherein said platinum compound is carboplatin.
 10. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of antibody-albumin nanoparticle complexes, said antibody-albumin nanoparticle complexes comprising albumin, an anti-CD52 antibody, and a therapeutically effective amount of paclitaxel, wherein the nanoparticle complexes have been pre-formed in vitro by mixing aqueous albumin-paclitaxel nanoparticles with the antibody under conditions to form the nanoparticle complexes, such that the antibody retains anti-CD52 binding specificity, wherein the average diameter of at least 90 percent of said complexes is less than 1
 11. The pharmaceutical composition of claim 10, wherein the antibody is a humanized antibody.
 12. The pharmaceutical composition of claim 10, wherein the antibody binds an antigen on a tumor cell.
 13. The pharmaceutical composition of claim 12, wherein the tumor cell is a solid tumor cell.
 14. The pharmaceutical composition of claim 12, wherein the tumor cell is derived from a lymphoma or a leukemia.
 15. The pharmaceutical composition of claim 12, wherein said composition or said nanoparticle complexes comprise an alkylating agent.
 16. The pharmaceutical composition of claim 15, wherein said alkylating agent is a platinum compound.
 17. The pharmaceutical composition of claim 16, wherein said platinum compound is carboplatin. 